Use of transthyretin peptide/protein fussions to increase the serum half-life of pharmacologically active peptides/proteins

ABSTRACT

The present invention provides a means for increasing the serum half-life of a selected biologically active agent by utilizing transthyretin (TTR) as a fusion partner with a biologically active agent. Specifically, the present invention provides substantially homogenous preparations of TTR (or a TTR variant)-biologically active agent fusions and PEG-TTR (PEG-TTR variant)-biologically active agent fusions. As compared to the biologically active agent alone, the TTR-biologically active agent fusion and/or PEG-TTR-biologically active agent fusion has substantially increased serum half-life.

This application is a Continuation of U.S. application Ser. No.10/407,078, filed Apr. 3, 2003, which is a Continuation-in-part of U.S.application Ser. No. 10/117,109, filed 04 Apr. 2002, which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Proteins, peptides and other drug molecules for therapeutic use arecurrently available in suitable forms in adequate quantities largely asa result of the advances in recombinant DNA technologies. Theavailability of such peptides and proteins has engendered advances inprotein formulation and chemical modification. Chemical modification ofbiologically active peptides, proteins, oligonucleotides and other drugsfor purposes of extending the serum half-life of such bioactive agentshas been extensively studied. The ability to extend the serum half-lifeof such agents allows for the therapeutic potential of the agent to berealized without the need for high dosages and frequent administration.

Chemical modification used to extend the half-lives of proteins in vivoincludes the chemical conjugation of a water soluble polymer, such aspolyethylene glycol (PEG), to the protein of interest. A variety ofapproaches have been used to attach the polyethylene glycol molecules tothe protein (PEGylation). For example, Royer (U.S. Pat. No. 4,002,531)states that reductive alkylation was used for attachment of polyethyleneglycol molecules to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337)disclose PEG:protein conjugates involving, for example, enzymes andinsulin. Shaw (U.S. Pat. No. 4,904,584) disclose the modification of thenumber of lysine residues in proteins for the attachment of polyethyleneglycol molecules via reactive amine groups. Hakimi et al. (U.S. Pat. No.5,834,594) disclose substantially non-immunogenic water solublePEG:protein conjugates, involving for example, the proteins IL-2,interferon alpha, and IL-1ra. The methods of Hakimi et al. involve theutilization of unique linkers to connect the various free amino groupsin the protein to PEG. Kinstler et al. (U.S. Pat. Nos. 5,824,784 and5,985,265) teach methods allowing for selectively N-terminallychemically modified proteins and analogs thereof, including G-CSF andconsensus interferon.

Other approaches designed to extend the serum half-life of bioactiveagents include: conjugation of the peptides to a large, stable proteinwhich is too large to be filtered through the kidneys (e.g., serumalbumin); G. D. Mao et al., Biomat., Art. Cells, Art. Org. 17:229-244(1989); use of low- and high-density lipoproteins as transport vehiclesand to increase serum half-life; P. Chris de Smidt et al., Nuc. Acids.Res., 19(17):4695-4700 (1991); the use of the Fc region ofimmunoglobulins to produce Fc-protein fusions; PCT WO 98/28427 (Mann etal, and references cited therein); and the use of the Fc domain toincrease in vivo half-life of one or more biologically active peptides;PCT WO 00/24782 (Feige et al, and references cited therein).

Transthyretin (TTR) (formerly called prealbumin) is a 56 kDa tetramericserum protein that plays important physiological roles as a transporterof thyroxine and retinol-binding protein; Hamilton and Benson, Cell.Mol. Life Sci., 58:1491-1521 (2001), and references cited therein.Blaney et al., in U.S. Pat. No. 5,714,142, describe the exploitation ofTTR by endowing the drug to be administered with functionality thatallows it to bind specifically to the protein. Specifically, Blaney etal. demonstrate that covalent attachment of a peptide, protein,nucleotide, oligonucleotide, oligosaccharide or other drug to atransthyretin-selective ligand will reversibly bind the drug to TTR andthereby increase the serum half-life of the agent based on the affinityof the ligand for TTR. It is stated that the intrinsic activity of thedrug is not adversely affected and the resulting drug-TTR ligandconjugate will still be small enough to be orally absorbed.

SUMMARY OF THE INVENTION

It has been found, surprisingly and importantly, that TTR (or a TTRvariant), and in particular, a TTR or TTR variant which has beenchemically modified via conjugation to a water soluble polymer, e.g.,can be used as a fusion partner with a biologically active agent toincrease the serum half-life of the biologically active agent.Accordingly, the present invention provides a means for increasing theserum half-life of a selected biologically active agent.

The present invention thus relates to substantially homogenouspreparations of TTR (or a TTR variant)-biologically active agent fusionsand PEG-TTR (PEG-TTR variant)-biologically active agent fusions. Ascompared to the biologically active agent alone, the TTR-biologicallyactive agent fusion and/or PEG-TTR-biologically active agent fusion hassubstantially increased serum half-life.

The present invention further relates to TTR-biologically active agentfusions and PEG-TTR-biologically active agent fusions, in apharmaceutically acceptable carrier, to provide a pharmacologicallyactive compound.

The present invention further relates to the preparation of TTRvariants. Specifically, TTR proteins are modified such that cysteineresidue(s) are engineered into the TTR protein sequence. The TTRvariants are recoverable in high yield and are then chemically modifiedvia conjugation of a water soluble polymer at the cysteine residue toprovide a chemically modified TTR variant which can then be fused to aselected biologically active agent.

The present invention further relates to processes for preparingpharmacologically active compounds. For example, the principalembodiment of the method for making the substantially homogenouspreparation of a PEG-TTR-peptide fusion comprises: (a) engineering acysteine residue into a specific amino acid position within the aminoacid sequence of said TTR to provide a variant of said TTR; (b)conjugating a polyethylene glycol to said TTR variant at said cysteineresidue to provide a PEG-TTR; (c) fusing said PEG-TTR to a peptide ofinterest to provide a PEG-TTR-peptide fusion; and (d) isolating saidPEG-TTR-peptide fusion.

The present invention also relates to methods of treatment ofindividuals using the pharmacologically active compounds as above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS gel that depicts the purification of an E. coliexpressed, recombinant human transthyretin (TTR) variant (C10A/G83C)with a Bradykinin peptide fused to the C-terminus of TTR. Lane 1contains NOVEX® 12 molecular weight standards, and lanes 2-7 contain thefollowing respectively: cell lysate, post-heating supernatant, pool fromQ-sepharose chromatography step, pool from phenyl sepharosechromatography step, pool from hydroxyapatite chromatography step, andpool from source Q chromatography step.

FIG. 2 demonstrates by size exclusion chromatography that fusion ofpeptides to the amino-terminus or carboxy-terminus of a TTR variant,TTR(C10A/G83C), does not alter its oligomeric structure. Solid line isTTR(C10A/G83C), dashed line is parathyroid hormone (PTH) fused to theamino-terminus of TTR(C10A/G83C), and the dotted line is Bradykininfused to the carboxy-terminus of TTR(C10A/G83C).

FIG. 3 demonstrates by size exclusion chromatography that fusion ofproteins to the amino-terminus or carboxy-terminus of a TTR variant,TTR(C10A), does not alter its oligomeric structure. Solid line isTTR(C10A), dashed line is IL-1-ra fused to the carboxy-terminus ofTTR(C10A), and the dotted line is IL-1-ra fused to the amino-terminus ofTTR(C10A).

FIG. 4 shows the binding observed using BIAcore of various TPO-mimeticpeptide (TMP) constructs to human MPL receptor: ▪ Fc-TMP,  TMP(m)-TTR,▴ TMP(m)-TTR-PEG5K, ▾ TMP(m)-TTR-PEG20K.

FIG. 5 shows that injection of TMP(m)-TTR-PEG5K induces plateletformation in mice. The following symbols correspond to the correspondingconstructs: ▪ Carrier,  Fc-TMP, ▴ TTR-TMP, ▾ TMP(m)-TTR, and ♦TMP(m)-TTR-PEG5K.

FIG. 6 demonstrates by size exclusion chromatography that native TTR andTTR(C10A) maintain a similar oligomeric configuration (an apparenttetramer). Solid line is native TTR and the dashed line is TTR(C10A).

FIG. 7 demonstrates by size exclusion chromatography that conjugation ofPEG to TTR increases its molecular size in a predictable uniform manner.Solid lines indicate no PEG conjugated, dashed lines indicate 5 K PEGfused, and dotted lines indicate 20 K PEG fused. The followingconstructs were used: A) TMP-TTR(C10A/A37C), B) TMP-TTR(C10A/D38C), C)TMP-TTR(C10A/A81C), and D) TMP-TTR(C10A/G83C).

FIG. 8 is an SDS gel that depicts the extent of pegylation of variousTMP-TTR constructs involving TTR variants having a non-native cysteineengineered in at one of four different locations. Lane 1 contains NOVEX®12 molecular weight standards; lane 2 is unpegylated TMP-TTR(C10A/A37C);lanes 3-6 are 5 K pegylated versions of TMP-TTR(C10A/A37C),TMP-TTR(C10A/D38C), TMP-TTR(C10A/A81C), and TMP-TTR(C10A/G83C)respectively; lanes 7-10 are 20 K pegylated versions ofTMP-TTR(C10A/A37C), TMP-TTR(C10A/D38C), TMP-TTR(C10A/A81C), andTMP-TTR(C10A/G83C), respectively.

FIGS. 9A-C compare the competitive binding of Fc-TMP and TMP-TTR tohuman MPL by BIAcore analysis. A) ▪ Fc-TMP,  TMP-TTR(C10A/A37C), ▴TMP-TTR(C10A/D38C), ▾ TMP-TTR(C10A/A81C), ♦ TMP-TTR(C10A/G83C). B) ▪Fc-TMP, 5 K pegylated versions of TMP-TTR(C10A/A37C) (),TMP-TTR(C10A/D38C) (▴), TMP-TTR(C10A/A81C) (▾), TMP-TTR(C10A/G83C)(♦).C) ▪ Fc-TMP, 20 K pegylated versions of TMP-TTR(C10A/A37C) (),TMP-TTR(C10A/D38C)(▴), TMP-TTR(C10A/A81C) (▾), TMP-TTR(C10A/G83C)(♦).

FIGS. 10A-C show that injection of TMP-TTR with PEG conjugated toengineered cysteines induces platelet formation in mice. A) ▪ TTR(C10A), Fc-TMP, ▴ TMP-TTR(C10A/A37C), ▾ TMP-TTR(C10A/D38C)(carboxamidomethylated), ♦ TMP-TTR(C10A/A81C),

TMP-TTR(C10A/G83C). B) ▪ TTR(C10A),  Fc-TMP, 5 K pegylated versions ofTMP-TTR(C10A/A37C)(▴), TMP-TTR(C10A/D38C) (▾), TMP-TTR(C10A/A81C)(♦),TMP-TTR(C10A/G83C)(

). C) ▪ TTR(C10A),  Fc-TMP, 20 K pegylated versions ofTMP-TTR(C10A/A37C) (▴), TMP-TTR(C10A/D38C) (▾), TMP-TTR(C10A/A81C)(♦),TMP-TTR(C10A/G83C)(

).

FIG. 11 shows that injection of PTH-TTR with PEG conjugated toengineered cysteines induces ionized calcium release in mice. Thefollowing symbols correspond to the corresponding constructs: ▪TTR(C10A),  PTH-Fc, ▴ PTH-TTR, ▾ PTH-TTR(C10A/K15A/A37C)(carboxamidomethylated), ♦ 5 K pegylated version ofPTH-TTR(C10A/K15A/A37C),

20 K pegylated version of PTH-TTR(C10A/K15A/A37C),

PTH-TTR(C10A/K15A/G83C) (carboxamidomethylated),

5 K pegylated version of PTH-TTR(C10A/K15A/G83C), and

20 K pegylated version of PTH-TTR(C10A/K15A/G83C).

FIG. 12 shows that injection of Glucagon-like Peptide 1 (GLP1)-TTR withPEG conjugated to engineered cysteines lowers blood glucose levels inmice. The following symbols correspond to the corresponding constructs:▪ TTR(C10A),  GLP1-Fc, ▴ GLP1-TTR(C10A/K15A/G83C) (PEG 5 K), and 0▾GLP1-TTR(C10A/K15A/G83C) (PEG 20 K).

FIG. 13 shows that injection of TMP-TTR conjugates with fused CH2domains increase serum platelet levels in mice. The following symbolscorrespond to the corresponding constructs: ▪ TTR(C10A),  Fc-TMP, ▴TMP-TTR(C10A)-CH2, ▾ TTR(C10A)-CH2-TMP, and ♦ TMP-CH2-TTR(C10A).

FIG. 14 shows that injection of and carboxy-terminal fusions of TMP withpegylated TTR increases blood platelet counts in mice. The followingsymbols correspond to the corresponding constructs: ▪ TTR(C10A), Fc-TMP, ▴ TTR(C10A/K15A/A37C)-TMP (PEG 20 K), ▾ TTR(C10A/K15A/A81C)-TMP(PEG 20 K), ♦ TTR(C10A/K15A/G83C)-TMP (PEG 20 K),

TMP-TTR(C10A/K15A/A37C) (PEG 20 K),

TMP-TTR(C10A/K15A/A81C) (PEG 20 K),

TMP-TTR(C10A/K15A/G83C) (PEG 20 K).

FIGS. 15 A-C show that injection of pegylated TMP-TTR fusions containinga K15A alteration increases blood platelet counts in mice. The followingsymbols correspond to the corresponding constructs: A) ▪ TTR(C10A), Fc-TMP, ▴ TMP-TTR(C10A/K15A/A37C) (carboxyamidomethylated), and ▾TMP-TTR(C10A/K15A/A81C) (carboxyamidomethylated); B) ▪ TTR(C10A), Fc-TMP, ▴ TMP-TTR(C10A/K15A/A37C) (PEG 5 K), ▾ TMP-TTR(C10A/K15A/A81C)(PEG 5 K), and ♦ TMP-TTR(C10A/K15A/G83C) (PEG 5 K); C) ▪ TTR(C10A), Fc-TMP, ▴ TMP-TTR(C10A/K15A/A37C) (PEG 20 K), ▾ TMP-TTR(C10A/K15A/A81C)(PEG 20 K), and ♦ TMP-TTR(C10A/K15A/G83C) (PEG 20 K).

DETAILED DESCRIPTION OF THE INVENTION

For purposes of describing the present invention, the following termsare defined as set forth below.

The term “biologically active agent” refers to any chemical material orcompound useful for prophylactic, therapeutic or diagnostic application.The term “pharmacologically active compound” refers to a compoundsuitable for administration to a mammalian, preferably a humanindividual, which induces a desired local or systemic effect.

The terms “peptide”, “polypeptide” and “protein” describe a type ofbiologically active agents, and the terms are used interchangeablyherein to refer to a naturally occurring, recombinantly produced orchemically synthesized polymer of amino acids. The terms are intended toinclude peptide molecules containing as few as 2 amino acids, chemicallymodified polypeptides, consensus molecules, analogs, derivatives orcombinations thereof.

Any number of peptides may be used in conjunction with the presentinvention. Of particular interest are peptides that mimic the activityof erythropoietin (EPO), thrombopoietin (TPO), Glucagon-like Peptide 1(GLP-1), parathyroid hormone (PTH), granulocyte-colony stimulatingfactor (G-CSF), granulocyte macrophage-colony stimulating factor(GM-CSF), interleukin-1 receptor antagonist (IL-1ra), leptin, cytotoxicT-lymphocyte antigen 4 (CTLA4), TNF-related apoptosis-inducing ligand(TRAIL), tumor growth factor-alpha and beta (TGF-α and TGF-β,respectively), and growth hormones. The terms “-mimetic peptide” and“-agonist peptide” refer to a peptide having biological activitycomparable to a protein (e.g., GLP-1, PTH, EPO, TPO, G-CSF, etc.) thatinteracts with a protein of interest. These terms further includepeptides that indirectly mimic the activity of a protein of interest,such as by potentiating the effects of the natural ligand of the proteinof interest. Thus, the term “EPO-mimetic peptide” comprises any peptidesthat can be identified or derived as having EPO-mimetic subject matter;see, for example, Wrighton et al., Science, 273:458-63 (1996); andNaranda et al., Proc. Natl. Acad. Sci. USA 96:7569-74 (1999). Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The term “TPO-mimetic peptide” (TMP) comprises peptides that can beidentified or derived as having TPO-mimetic subject matter; see, forexample, Cwirla et al., Science, 276:1696-9 (1997); U.S. Pat. Nos.5,869,451 and 5,932,946; and PCT WO 00/24782 (Liu et al, and referencescited therein), hereby incorporated by reference in its entirety. Thoseof ordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The term “G-CSF-mimetic peptide” comprises any peptides that can beidentified as having G-CSF-mimetic subject matter; see, for example,Paukovits et al., Hoppe-Seylers Z. Physiol. Chem. 365:303-11 (1984).Those of ordinary skill in the art appreciate that each of thesereferences enables one to select different peptides than actuallydisclosed therein by following the disclosed procedures with differentpeptide libraries.

The term “CTLA4-mimetic peptide” comprises any peptides that can beidentified or derived as described in Fukumoto et al., Nature Biotech.16:267-70 (1998). Those of ordinary skill in the art appreciate thateach of these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

Peptide antagonists are also of interest, particularly thoseantagonistic to the activity of TNF, leptin, any of the interleukins,and proteins involved in complement activation (e.g., C3b). The term“-antagonist peptide” or “inhibitor peptide” refers to a peptide thatblocks or in some way interferes with the biological activity of theassociated protein of interest, or has biological activity comparable toa known antagonist or inhibitor of the associated protein of interest.Thus, the term “TNF-antagonist peptide” comprises peptides that can beidentified or derived as having TNF-antagonistic subject matter; see,foe example, Takasaki et al., Nature Biotech., 15:1266-70 (1997). Thoseof ordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The terms “IL-1 antagonist” and “IL-1ra-mimetic peptide” comprisespeptides that inhibit or down-regulate activation of the IL-1 receptorby IL-1. IL-1 receptor activation results from formation of a complexamong IL-1, IL-1 receptor, and IL-1 receptor accessory protein. IL-1antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor, orIL-1 receptor accessory protein and obstruct complex formation among anytwo or three components of the complex. Exemplary IL-1 antagonist orIL-1ra-mimetic peptides can be identified or derived as described inU.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096. Those of ordinary skillin the art appreciate that each of these references enables one toselect different peptides than actually disclosed therein by followingthe disclosed procedures with different peptide libraries.

The term “VEGF-antagonist peptide” comprises peptides that can beidentified or derived as having VEGF-antagonistic subject matter; see,for example, Fairbrother, Biochem., 37:17754-64 (1998). Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The term “MMP inhibitor peptide” comprises peptides that can beidentified or derived as having MMP inhibitory subject matter; see, forexample, Koivunen, Nature Biotech., 17:768-74 (1999). Those of ordinaryskill in the art appreciate that each of these references enables one toselect different peptides than actually disclosed therein by followingthe disclosed procedures with different peptide libraries.

Targeting peptides are also of interest, including tumor-homingpeptides, membrane-transporting peptides, and the like.

Exemplary peptides may be randomly generated by various techniques knownin the art. For example, solid phase synthesis techniques are well knownin the art, and include those described in Merrifield, Chem.Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.)(1973);Merrifield, J. Am. Chem. Soc., 85:2149 (1963); Davis et al., Biochem.Intl., 10:394-414 (1985); Stewart and Young, Solid Phase PeptideSynthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., The Proteins,3rd ed., 2:105-253 (1976); and Erickson et al., The Proteins, 3rd ed.,2:257-527 (1976). Solid phase synthesis is the preferred technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides.

Phage display is another useful method in generating peptides for use inthe present invention. It has been stated that affinity selection fromlibraries of random peptides can be used to identify peptide ligands forany site of any gene product; Dedman et al., J. Biol. Chem.,268:23025-30 (1993). Phage display is particularly well suited foridentifying peptides that bind to such proteins of interest as cellsurface receptors or any proteins having linear epitopes; Wilson et al.,Can. J. Microbiol., 44:313-29 (1998); Kay et al., Drug Disc. Today,3:370-8 (1998). Such proteins are extensively reviewed in Herz et al.,J. Receptor & Signal Transduction Res., 17(5):671-776 (1997), which ishereby incorporated by reference.

The peptides may also be made in transformed host cells usingrecombinant DNA techniques. To do so, a recombinant DNA molecule codingfor the peptide is prepared. Methods of preparing such DNA and/or RNAmolecules are well known in the art. For instance, sequences coding forthe peptides could be excised from DNA using suitable restrictionenzymes. The relevant sequences can be created using the polymerasechain reaction (PCR) with the inclusion of useful restriction sites forsubsequent cloning. Alternatively, the DNA/RNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidite method. Also, a combination of these techniques could beused.

Additional biologically active agents contemplated for use includerecombinant or naturally occurring proteins, whether human or animal,hormones, cytokines, hematopoietic factors, growth factors, antiobesityfactors, trophic factors, anti-inflammatory factors, and enzymes. Suchproteins would include but are not limited to interferons (see, U.S.Pat. Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 herebyincorporated by reference including drawings), interleukins (see, U.S.Pat. No. 5,075,222, hereby incorporated by reference includingdrawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,5,618,698 5,547,933, and 5,621,080 hereby incorporated by referenceincluding drawings), granulocyte-colony stimulating factors (see, U.S.Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCTPublication No. 94/17185, hereby incorporated by reference includingdrawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505,and 95/17206, hereby incorporated by reference including drawings),novel erythropoietin stimulating protein (NESP) (PCT Publication No.US94/09257, hereby incorporated by reference including drawings),osteoprotegerin (PCT Publication No. 97/23614, hereby incorporated byreference including drawings), interleukin-1 receptor antagonist(IL-1ra)(PCT Publication Nos. 91/08285 and 92/16221) and leptin (OBprotein) (PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010and 97/06816 hereby incorporated by reference including figures).

In addition, biologically active agents can also include but are notlimited to insulin, gastrin, prolactin, adrenocorticotropic hormone(ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH),follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG),motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12),tumor necrosis factor (TNF), tumor necrosis factor-binding protein(TNF-bp), brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblastgrowth factors (FGF), neurotrophic growth factor (NGF), bone growthfactors such as osteoprotegerin (OPG), insulin-like growth factors(IGFs), macrophage colony stimulating factor (M-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), megakaryocyte derivedgrowth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin,platelet-derived growth factor (PGDF), colony simulating growth factors(CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD),tissue plasminogen activator (TPA), urokinase, streptokinase andkallikrein.

Transthyretin (TTR) contemplated for use in the present invention willhave the DNA and amino acid sequences of TTR as reported in Mita et al.,Biochem. Biophys. Res. Commun., 124(2):558-564 (1984). These sequenceshave been deposited in Genbank as accession number K02091. The 127 aminoacid TTR sequence used herein does not include the signal sequence(amino acids 1-20) of the K02091 sequence and is depicted below as SEQID NO:1.

SEQ ID NO:1 GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE

The term “TTR variant” refers to a molecule or sequence that is amodified form of a native TTR. For example, a native TTR comprises sitesthat may be removed because they provide structural features orbiological activity that are not required for the fusion molecules ofthe present invention. Thus, the term “TTR variant” comprises a moleculeor sequence that lacks one or more native TTR sites or residues or thathas had one or more native TTR sites or residues replaced with adifferent amino acid or that has had one or more residues added to thesequence. For purposes of an example, a TTR variant wherein the Alanineresidue at amino acid sequence position 37 has been replaced with aCysteine residue, will be designated TTR variant (A37C); and a TTRvariant wherein both the Alanine residue at amino acid sequence position37 and the Glycine residue at amino acid sequence position 83 have bothbeen replaced with a Cysteine residue will be designated TTR variant(A37C/G83C).

In one embodiment, a TTR or TTR variant fused to a biologically activeagent may be fused to a third protein or protein fragment that furtherstabilizes the TTR-biologically active agent fusion protein, and therebyincreases the half-life of the resulting fusion in serum. Examples ofsuch additional proteins or fragments thereof that can be used in suchmethods and compositions include the Fc domain or CH2 domain of animmunoglobulin, or any other protein domain that one of skill in the artwould recognize as having properties that would increase proteinstability (see, e.g., Example 29 below). Such protein groups can befused to the carboxy or amino terminus of the TTR-biologically activeagent fusion protein, or can be placed between the TTR and thebiologically active agent. It is contemplated that linkers or spacerscan be placed, as needed, between each of the domains of the fusionprotein to facilitate their desired activity.

In another embodiment, the TTR or TTR variant of the invention can bechemically crosslinked to the biologically active agent. Cross-linkingof proteins can be performed by using, for example, N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) according to established,published procedures. Additional cross-linking agents are readilyavailable and can be identified by one of skill in the art. For detailson the above procedure, see, e.g., Karpovsky et al, J. Exp. Med. 160,1686-1701 (1984); Perez et al, Nature, 316, 354-356 (1985) or Titus etal, Journal of Immunology, 139, 3153-3158 (1987).

In another embodiment, a molecule can be covalently linked to the fusionprotein such that stability and/or half-life in serum are increased. Forexample, a preferred TTR or TTR variant may be chemically modified usingwater soluble polymers such as polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be straight chain orbranched. The average molecular weight of the PEG will preferably rangefrom about 2 kDa to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably about 20 kDa.

The PEG groups will generally be attached to the compounds of theinvention via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the peg moiety (e.g., an aldehyde, amino, ester,thiol, -haloacetyl, maleimido or hydrazino group) to a reactive group onthe target compound (e.g., an aldehyde, amino, ester, thiol,-haloacetyl, maleimido or hydrazino group).

Other water soluble polymers used include copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, polyvinyl alcohol,polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran.

A DNA molecule encoding the peptide of interest, protein of interest,TTR or TTR variant can be prepared using well known recombinant DNAtechnology methods such as those set forth in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. [1989]) and/or Ausubel et al., eds, CurrentProtocols in Molecular Biology, Green Publishers Inc. and Wiley andSons, NY (1994). A gene or cDNA encoding the protein of interest orfragment thereof may be obtained for example by screening a genomic orcDNA library with a suitable probe. Suitable probes include, forexample, oligonucleotides, cDNA fragments, or genomic DNA fragments,that are expected to have some homology to the gene encoding the proteinof interest, such that the probe will hybridize with the gene encodingthe protein of interest under selected hybridization conditions. Analternate means of screening a DNA library is by polymerase chainreaction “PCR” amplification of the gene encoding the protein ofinterest. PCR is typically accomplished using oligonucleotide “primers”which have a sequence that is believed to have sufficient homology tothe gene to be amplified such that at least a sufficient portion of theprimer will hybridize with the gene.

Alternatively, a gene encoding the peptide of interest or protein ofinterest may be prepared by chemical synthesis using methods well knownto the skilled artisan such as those described by Engels et al., Angew.Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia,the phosphotriester, phosphoramidite, and H-phosphonate methods fornucleic acid synthesis. A preferred method for such chemical synthesisis polymer-supported synthesis using standard phosphoramidite chemistry.Typically, the DNA encoding the protein of interest will be severalhundred nucleotides in length. Nucleic acids larger than about 100nucleotides can be synthesized as several fragments using these methods.The fragments can then be ligated together to form a gene coding for thefull length protein of interest. Usually, the DNA fragment encoding theamino terminus of the polypeptide will have an ATG, which encodes amethionine residue. This methionine may or may not be present on themature form of the protein of interest. The methionine can be removedinside the cell or during the process of secretion. Preferred TTRpolypeptides may include TTR with the nucleic acid sequence altered tooptimize expression in E. coli and to introduce convenient restrictionsites. A general discussion of codon optimization for expression in E.coli is described in Kane, Curr. Opin. Biotechnol., 6:494-500 (1995).

Once the genes encoding the protein of interest and the TTR polypeptidehave been obtained, they may be modified using standard methods tocreate restriction endonuclease sites at the 5′ and/or 3′ ends. Creationof the restriction sites permits the genes to be properly inserted intoamplification and/or expression vectors. Addition of restriction sitesis typically accomplished using PCR, where one primer of each PCRreaction typically contains, inter alia, the nucleotide sequence of thedesired restriction site.

The gene or cDNA encoding the peptide of interest, or protein ofinterest can be inserted into an appropriate expression vector forexpression in a host cell. The vector is selected to be functional inthe particular host cell employed (i.e., the vector is compatible withthe host cell machinery such that amplification and/or expression of thegene encoding the protein of interest can occur).

Typically, the vectors used in any of the host cells will contain apromoter (also referred to as a “5′ flanking sequence”) and otherregulatory elements as well such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a ribosomebinding site element, a polylinker region for inserting the nucleic acidencoding the polypeptide to be expressed, and a selectable markerelement. Each of these elements is discussed below. Optionally, thevector may contain a “tag” DNA sequence, i.e., an oligonucleotidesequence located at either the 5′ or 3′ end of the fusion DNA construct.The tag DNA encodes a molecule such as hexaHis, c-myc, FLAG (Invitrogen,San Diego, Calif.) or another small immunogenic sequence. When placed inthe proper reading frame, this tag will be expressed along with thefusion protein, and can serve as an affinity tag for purification of thefusion protein from the host cell. Optionally, the tag can subsequentlybe removed from the purified fusion protein by various means such asusing a selected peptidase for example.

The promoter may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination ofpromoters from more than one source), synthetic, or it may be the nativeprotein of interest promoter. Further, the promoter may be aconstitutive or an inducible promoter. As such, the source of thepromoter may be any unicellular prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that thepromoter is functional in, and can be activated by, the host cellmachinery.

The promoters useful in the vectors of this invention may be obtained byany of several methods well known in the art. Typically, promotersuseful herein will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of the promoter may be known.Here, the promoter may be synthesized using the methods described abovefor nucleic acid synthesis or cloning.

Where all or only a portion of the promoter sequence is known, thecomplete promoter may be obtained using PCR and/or by screening agenomic library with suitable oligonucleotide and/or 5′ flankingsequence fragments from the same or another species.

Suitable promoters for practicing this invention are inducible promoterssuch as the lux promoter, the lac promoter, the arabinose promoter, thetrp promoter, the tac promoter, the tna promoter, synthetic lambdapromoters (from bacteriophage lambda), and the T5 or T7 promoters.Preferred promoters include the lux, and lac promoters.

The origin of replication element is typically a part of prokaryoticexpression vectors whether purchased commercially or constructed by theuser. In some cases, amplification of the vector to a certain copynumber can be important for optimal expression of the protein orpolypeptide of interest. In other cases, a constant copy number ispreferred. In any case, a vector with an origin of replication thatfulfills the requirements can be readily selected by the skilledartisan. If the vector of choice does not contain an origin ofreplication site, one may be chemically synthesized based on a knownsequence, and ligated into the vector.

The transcription termination element is typically located 3′ of the endof the fusion protein DNA construct, and serves to terminatetranscription of the RNA message coding for the fusion polypeptide.Usually, the transcription termination element in prokaryotic cells is aG-C rich fragment followed by a poly T sequence. While the element iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described above.

Expression vectors typically contain a gene coding for a selectablemarker. This gene encodes a protein necessary for the survival andgrowth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline,chloramphenicol, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, thechloramphenicol resistance gene, and the tetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence in prokaryotes, is necessary for the initiation of translationof mRNA. The element is typically located 3′ to the promoter and 5′ tothe coding sequence of the fusion protein DNA construct. TheShine-Dalgarno sequence is varied but is typically a polypurine (i.e.,having a high A-G content). Many Shine-Dalgarno sequences have beenidentified, each of which can be readily synthesized using methods setforth above and used in a prokaryotic vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

Each element may be individually ligated into the vector by cutting thevector with the appropriate restriction endonuclease(s) such that theends of the element to be ligated in and the ends of the vector arecompatible for ligation. In some cases, it may be necessary to “blunt”the ends to be ligated together in order to obtain a satisfactoryligation. Blunting can be accomplished by first filling in “sticky ends”using an enzyme such as Klenow DNA polymerase or T4 DNA polymerase inthe presence of all four nucleotides. This procedure is well known inthe art and is described for example in Sambrook et al., supra.

Alternatively, two or more of the elements to be inserted into thevector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

Another method for constructing the vector is to conduct all ligationsof the various elements simultaneously in one reaction mixture. Here,many nonsense or nonfunctional vectors may be generated due to improperligation or insertion of the elements, however the functional vector maybe identified by expression of the selectable marker. Proper sequence ofthe ligation product can be confirmed by digestion with restrictionendonucleases or by DNA sequencing.

After the vector has been constructed and a fusion protein DNA constructhas been inserted into the proper site of the vector, the completedvector may be inserted into a suitable host cell for fusion proteinexpression.

Host cells suitable for the present invention are bacterial cells. Forexample, the various strains of E. coli (e.g., HB101, JM109, DH5α, DH10,and MC1061) are well-known host cells for use in preparing recombinantpolypeptides. The choice of bacterial strain is typically made so thatthe strain and the expression vector to be used are compatible. Variousstrains of B. subtilis, Pseudomonas spp., other Bacillus spp.,Streptomyces spp., and the like may also be employed in practicing thisinvention in conjunction with appropriate expression vectors.

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using suchmethods as calcium phosphate precipitation or electroporation. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,supra.

The host cells containing the vector (i.e., transformed or transfectedhost cells) may be cultured using one or more standard media well knownto the skilled artisan. The selected medium will typically contain allnutrients necessary for the growth and survival of the host cells.Suitable media for culturing E. coli cells, are, for example, Luriabroth (“LB”), YT broth, SOB, SOC, and/or Terrific Broth (“TB”).

There are several ways to prepare the DNA construct encoding the fusionprotein which comprises the TTR gene, the gene encoding the peptide orprotein of interest, and, optionally, a DNA molecule encoding a linkerpeptide which is located between the two genes.

In one procedure, the TTR gene and gene encoding the protein of interest(the “fusion partner genes”) can be ligated together in eitherorientation (e.g., TTR gene at the 5′ or 3′ end of the construct). Wherea linker DNA molecule is to be included, it can first be ligated to oneof the fusion partner genes, and that construct can then be ligated tothe other fusion partner gene. Ligations are typically accomplishedusing DNA ligase enzyme in accordance with the manufacturer'sinstructions.

A separate procedure provides for first ligating one fusion partner geneinto the selected vector, after which the other fusion partner gene canbe ligated into the vector in a position that is either 3′ or 5′ to thefirst fusion partner gene. Where a linker DNA molecule is to beincluded, the linker DNA molecule may be ligated to either fusionpartner gene either before or after that gene has been ligated into thevector.

The TTR-TMPs of the present invention can be used to treat conditionsgenerally known as those that involve an existing megakaryocyte/plateletdeficiency or an expected megakaryocyte/platelet deficiency (e.g.,because of planned surgery or platelet donation). Such conditions willusually be the result of a deficiency (temporary or permanent) of activeMpl ligand in vivo. The generic term for platelet deficiency isthrombocytopenia, and hence the methods and compositions of the presentinvention are generally available for treating thrombocytopenia inpatients in need thereof. Thrombocytopenia (platelet deficiencies) maybe present for various reasons, including chemotherapy and other therapywith a variety of drugs, radiation therapy, surgery, accidental bloodloss, and other specific disease conditions.

Exemplary specific disease conditions that involve thrombocytopenia andmay be treated in accordance with this invention are: aplastic anemia,idiopathic thrombocytopenia, metastatic tumors which result inthrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi'ssyndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglinanomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnalhemoglobinuria. Also, certain treatments for AIDS result inthrombocytopenia (e.g., AZT). Certain wound healing disorders might alsobenefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, a compound of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, acompound of this invention could be administered along with blood orpurified platelets.

The TMP compounds of this invention may also be useful in stimulatingcertain cell types other than megakaryocytes if such cells are found toexpress Mpl receptor. Conditions associated with such cells that expressthe Mpl receptor, which are responsive to stimulation by the Mpl ligand,are also within the scope of this invention.

The TMP compounds of this invention may be used in any situation inwhich production of platelets or platelet precursor cells is desired, orin which stimulation of the c-Mpl receptor is desired. Thus, forexample, the compounds of this invention may be used to treat anycondition in a mammal wherein there is a need of platelets,megakaryocytes, and the like. Such conditions are described in detail inthe following exemplary sources: WO95/26746; WO95/21919; WO95/18858;WO95/21920 and are incorporated herein.

The TMP compounds of this invention may also be useful in maintainingthe viability or storage life of platelets and/or megakaryocytes andrelated cells. Accordingly, it could be useful to include an effectiveamount of one or more such compounds in a composition containing suchcells.

The therapeutic methods, compositions and compounds of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl receptor, hematopoietic factors, interleukins,growth factors or antibodies in the treatment of disease statescharacterized by other symptoms as well as platelet deficiencies. It isanticipated that the inventive compound will prove useful in treatingsome forms of thrombocytopenia in combination with general stimulatorsof hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyticstimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemiainhibitory factor (LIF), oncostatin M (OSM), or other molecules withmegakaryocyte stimulating activity may also be employed with Mpl ligand.Additional exemplary cytokines or hematopoietic factors for suchco-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It mayfurther be useful to administer, either simultaneously or sequentially,an effective amount of a soluble mammalian Mpl receptor, which appearsto have an effect of causing megakaryocytes to fragment into plateletsonce the megakaryocytes have reached mature form. Thus, administrationof an inventive compound (to enhance the number of maturemegakaryocytes) followed by administration of the soluble Mpl receptor(to inactivate the ligand and allow the mature megakaryocytes to produceplatelets) is expected to be a particularly effective means ofstimulating platelet production. The appropriate dosage would beadjusted to compensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byconventional methods.

In non-insulin dependent diabetes mellitus (NIDDM), also known as type 2diabetic patients, the administration of glucagon-like peptide-1 (GLP-1)has antidiabetic properties. However, GLP-1 is rapidly degraded bydipeptidyl peptidase IV (DPPIV) after its release in vivo. Thus, it isan advantage of the present invention that a GLP-1 peptide or variantthereof can be fused to a TTR polypeptide of the invention to stabilizeGLP-1 and increase its half life in vivo. Accordingly, in anotherembodiment of the invention, a TTR-GLP1 fusion protein as describedherein can be used to treat conditions generally known to involvenon-insulin dependent diabetes mellitus (NIDDM), which is also known astype II diabetes.

One of skill in the art will recognize that the sequence of a GLP-1peptide can be varied such that it retains its insulinotropic effects.Particular examples of such variations known in the art include, forexample, GLP-1 (7-34), (7-35), (7-36) or (7-37), Gln⁹-GLP-1 (7-37),D-Gln⁹-GLP-1 (7-37), Thr¹⁶-Lys¹⁸-GLP-1 (7-37), and Lys¹⁸-GLP-1 (7-37).Additional examples of GLP-1 variants are described in U.S. Pat. Nos.5,118,666, 5,545,618, 5,977,071, and WO 02/46227 and in Adelhorst etal., J. Biol. Chem. 269:6275 (1994), which are incorporated byreference. Accordingly, any GLP-1 peptide can be used to generate fusionproteins of the invention, as long as the GLP-1 fusion protein iscapable of binding and inducing a signal through it's cognate receptor.Receptor binding and activation can be measured by standard assays (U.S.Pat. No. 5,120,712).

The dose of fusion protein effective to normalize a patient's bloodglucose will depend on a number of factors among which are included thesubject's weight, age, severity of their inability to regulate bloodglucose, the route of administration, the bioavailability, thepharmokinetic profile of the fusion protein and the formulation as isdiscussed more fully below.

The therapeutic methods, compositions and compounds of the presentinvention may also be employed, alone or in combination with otherdiabetes treatments, including but not limited to insulin,DPPIV-inhibitors and the like. The dosage of the GLP-1 fusion proteinwould be adjusted to compensate for such additional components in thetherapeutic composition. Progress of the treated patient can bemonitored by conventional methods, such as, for example, the monitoringof blood glucose levels.

The present invention also provides pharmaceutical compositions of theinventive compounds. Such pharmaceutical compositions may be foradministration for injection, or for oral, nasal, transdermal or otherforms of administration, including, e.g., by intravenous, intradermal,intramuscular, intramammary, intraperitoneal, intrathecal, intraocular,retrobulbar, intrapulmonary (e.g., aerosolized drugs) or subcutaneousinjection (including depot administration for long term release); bysublingual, anal, vaginal, or by surgical implantation, e.g., embeddedunder the splenic capsule, brain, or in the cornea. The treatment mayconsist of a single dose or a plurality of doses over a period of time.In general, comprehended by the invention are pharmaceuticalcompositions comprising effective amounts of a compound of the inventiontogether with pharmaceutically acceptable diluents, preservatives,stabilizers, solubilizers, emulsifiers, adjuvants and/or carriers. Suchcompositions include diluents of various buffer content (e.g., Tris-HCl,acetate, phosphate, citrate, etc.), pH and ionic strength; additivessuch as detergents and solubilizing agents (e.g., Tween 80, Polysorbate80, etc.), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances(e.g., lactose, mannitol); incorporation of the material intoparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also beused, and this may have the effect of promoting sustained duration inthe circulation. The pharmaceutical compositions optionally may includestill other pharmaceutically acceptable liquid, semisolid, or soliddiluents that serve as pharmaceutical vehicles, excipients, or media,including but are not limited to, polyoxyethylene sorbitan monolaurate,magnesium stearate, methyl- and propylhydroxybenzoate, starches,sucrose, dextrose, gum acacia, calcium phosphate, mineral oil, cocoabutter, and oil of theobroma. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilized form. Implantable sustained releaseformulations are also contemplated, as are transdermal formulations.

Controlled release formulation may be desirable. The drug could beincorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms e.g., gums. Slowly degeneratingmatrices may also be incorporated into the formulation, e.g., alginates,polysaccharides. Another form of a controlled release of thistherapeutic is by a method based on the Oros therapeutic system (AlzaCorp.), i.e., the drug is enclosed in a semipermeable membrane whichallows water to enter and push drug out through a single small openingdue to osmotic effects. Some enteric coatings also have a delayedrelease effect.

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei et al.,International Journal of Pharmaceutics 63:135-144 (1990)(leuprolideacetate); Braquet et al., Journal of Cardiovascular Pharmacology 13(suppl. 5): s.143-146 (1989)(endothelin-1); Hubbard et al., Annals ofInternal Medicine 3:206-212 (1989)(1-antitrypsin); Smith et al., J.Clin. Invest. 84:1145-1146 (1989)(1-proteinase); Oswein et al.,“Aerosolization of Proteins”, Proceedings of Symposium on RespiratoryDrug Delivery II, Keystone, Colo., March, 1990 (recombinant human growthhormone); Debs et al., The Journal of Immunology 140:3482-3488(1988)(interferon- and tumor necrosis factor ) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered dose inhalersand powder inhalers, all of which are familiar to those skilled in theart.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10μm (ormicrons), most preferably 0.5 to 5μm, for most effective delivery to thedistal lung.

Carriers include carbohydrates such as trehalose, mannitol, xylitol,sucrose, lactose, and sorbitol. Other ingredients for use informulations may include DPPC, DOPE, DSPC and DOPC. Natural or syntheticsurfactants may be used. Polyethylene glycol may be used (even apartfrom its use in derivatizing the protein or analog). Dextrans, such ascyclodextran, may be used. Bile salts and other related enhancers may beused. Cellulose and cellulose derivatives may be used. Amino acids maybe used, such as use in a buffer formulation.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the dose should be in the range of 0.1 μg to 100 mg of the inventivecompound per kilogram of body weight per day, preferably 0.1 to 1000μg/kg; and more preferably 0.1 to 150 μg/kg, given in daily doses or inequivalent doses at longer or shorter intervals, e.g., every other day,twice weekly, weekly, or twice or three times daily.

The inventive compounds may be administered by an initial bolus followedby a continuous infusion to maintain therapeutic circulating levels ofdrug product. As another example, the inventive compounds may beadministered as a one-time dose. Those of ordinary skill in the art willreadily optimize effective dosages and administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient. The frequency of dosing will depend on thepharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea or organ size.

Appropriate dosages may be ascertained through use of established assaysfor determining serum levels in conjunction with appropriatedose-response data. The final dosage regimen will be determined by theattending physician, considering various factors which modify the actionof drugs, e.g. the drug's specific activity, the severity of the damageand the responsiveness of the patient, the age, condition, body weight,sex and diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding the appropriate dosage levelsand duration of treatment for various diseases and conditions.

The following Examples are intended for illustration purposes only, andshould not be construed to limit the invention in any way.

Example 1

This example describes the preparation of DNA for native recombinanthuman transthyretin (TTR) and the following TTR variants: TTR(C10A),TTR(C10A/A37C), TTR(C10A/D38C), TTR(C10A/A81C), TTR(C10A/G83C), andTTR(C10A/K15A/G83C).

The expression plasmid pAMG21 is available from the ATCC under accessionnumber 98113, which was deposited on Jul. 24, 1996 (see PCT WO 97/23614,published 3 Jul. 1997 for a description of pAMG21). DNA sequence codingfor TTR, TTR variants or TTR-peptide fusions was placed under control ofthe LuxPR promoter in pAMG21.

The bacterial host GM221 is an E. coli K-12 strain that has beenmodified to contain both the temperature sensitive lambda repressorcI857s7 in the early ebg region and the lacI^(Q) repressor in the lateebg region (68 minutes). The presence of these two repressor genesallows the use of this host with a variety of expression systems,however both of these repressors are irrelevant to the expression fromluxP_(R). The untransformed host has no antibiotic resistances. Theribosomal binding site of the cI857s7 gene has been modified to includean enhanced RBS. It has been inserted into the ebg operon betweennucleotide position 1170 and 1411 as numbered in Genbank accessionnumber M64441Gb_Ba with deletion of the intervening ebg sequence. Theconstruct was delivered to the chromosome using a recombinant phagecalled MMebg-cI857s7 enhanced RBS #4 into F'tet/393. After recombinationand resolution only the chromosomal insert described above remains inthe cell. It was renamed F'tet/GM101. F'tet/GM101 was then modified bythe delivery of a lacI^(Q) construct into the ebg operon betweennucleotide position 2493 and 2937 as numbered in the Genbank accessionnumber M64441Gb_Ba with the deletion of the intervening ebg sequence.The construct was delivered to the chromosome using a recombinant phagecalled AGebg-lacI^(Q) #5 into F'tet/GM101. After recombination andresolution only the chromosomal insert described above remains in thecell. It was renamed F'tet/GM221. The F'tet episome was cured from thestrain using acridine orange at a concentration of 25 μg/ml in LB. Thecured strain was identified as tetracyline sensitive and was stored asGM221.

Oligonucleotides (1.0 nm each) were synthesized by phosphoramiditemethod. Nucleotides were, in some cases, altered for optimizedexpression in E. coli. These codon changes did not result in changes inthe amino acid sequence. Each of the oligonucleotides utilized in thisexample are listed in Table 1.

PCR was performed with the Expand Long Polymerase according to themanufacturer's protocol (Boehringer Mannheim). PCR products wereverified by agarose gel electrophoresis, purified and digested with Nde1and Xho1 (New England Biolabs). Expression vector pAMG21 was digested inthe same manner and then treated with calf intestinal phosphatase(Boehringer Mannheim). The vector and insert were purified from anagarose gel, then mixed and ligated by T4 DNA ligase (New EnglandBiolabs). Ligation was done at 4° C. for 2 hrs. Each ligation mixturewas transformed by electroporation into the host strain GM221 describedabove with a Biorad GenePulser (Biorad Laboratories) using 2.5V, 25 uFD,and 200 ohms in a cuvette with a gap length of about 2 mm. Afterelectroporation, the cells were allowed to recover in 1 ml of Luriabroth (LB) for about one hour at 37° C. with gentle shaking. The entiretransformation mix was plated on LB agar containing 50 ug/ml kanamycin.Colonies were screened for presence of the desired molecular weight byPCR using oligonucleotides directed against flanking vector sequence.The PCR products were evaluated by agarose gel electrophoresis. Positiveclones were further screened for the ability to produce the recombinantprotein product and finally verified by nucleotide sequencing.

The DNA and amino acid sequences of TTR are known (Mita, S et al.,Biochem. Biophys. Res. Commun. 124 (2), 558-564 [1984]). These sequenceshave been deposited in Genbank as accession number K02091. The cDNA ofnative TTR excluding the signal peptide was cloned from a cDNA libraryderived from human liver (Clontech). Specifically, an oligonucleotideencoding eight codons of the TTR 5′ (Oligo 2693-79) end and anoligonucleotide encoding seven codons of TTR 3′ end including aterminating codon (Oligo 2693-80) were synthesized and used to amplifythe full-length mature TTR with Expand Long polymerase using human livercDNA library as template. The resulting PCR fragment was digested withNdeI and XhoI, gel purified and ligated with NdeI/XhoI restrictedexpression vector pAMG21. After 2 hours at 4° C., the ligation mixturewas electroporated into GM221 cells. Single colonies were picked andplasmid DNA was prepared and sequenced. One resulting plasmid (strain#5316) was shown to have the correct DNA sequence of native TTR (plus amethionine at the N-terminus) and was used for expression. This DNAsequence is identified in SEQ ID NO:2.

Mutant TTR(C10A) was made by using oligonucleotide 2693-80 above andoligonucleotide 2820-88 (encompasses the first 11 codons of native TTRin which the codon Cys at the tenth position was changed to Ala). ThePCR procedure and the process for selecting the expression strain weresimilar to that described above. The resulting strain (strain #5619) hadthe DNA sequence identified in SEQ ID NO:3.

Plasmid 5619 was further modified by replacing the amino acids at thefollowing positions: A37, D38, A81 and G83, with the amino acidCysteine. As described below, each pair of the complementaryoligonucleotides harboring the desired mutations was used in conjunctionwith TTR 5′ and 3′ primers described above in a standard two-step PCRprocedure designed for site-specific mutagenesis. Each of the forwardprimers were used with a TTR 3′ primer and each of the reverse primerswere used with a TTR 5′ primer in a 20-cycle PCR in which plasmidderived from strain 5619 was used as the template. The resulting PCRamplified 5′ and 3′ fragments were mixed and used as the template forthe second step PCR to generate the full-length mutants. Subsequentcloning and sequencing procedures were similar to those alreadydescribed. The following oligonucleotides were utilized: TTR(A37C)forward (Oligo 2823-91); TTR(A37C) reverse (Oligo 2823-92); TTR(D38C)forward (Oligo 2823-93); TTR(D38C) reverse (Oligo 2823-94); TTR(A81C)forward (Oligo 2823-95); TTR(A81C) reverse (Oligo 2823-96); TTR(G83C)forward (Oligo 2823-97); TTR(G83C) reverse (Oligo 2823-98). Theresulting E. coli strains containing the plasmids are described asfollows: TTR(C10A/A37C)(strain 5641) had the DNA sequence identified inSEQ ID NO:4. TTR(C10A/D38C)(strain 5642) had the DNA sequence identifiedin SEQ ID NO:5. TTR(C10A/A81C)(strain 5643) had the DNA sequenceidentified in SEQ ID NO:6. TTR(C10A/G83C)(strain 5651) had the DNAsequence identified in SEQ ID NO:7.

The Lys in the 15th position in strain 5651 was further mutagenized toAla using oligonucleotides 2953-67 and 2953-68 by a procedure similar tothat described for strains 5641, 5642, 5643 and 5651.

The resulting strain, TTR(C10A/K15A/G83C)(strain 5895) had the DNAsequence identified in SEQ ID NO:8.

TABLE 1 SEQ ID Oligo Sequence Number 2693-79GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 18 2693-80CCGCGGATCCTCGAGATTATTCCTTGGGATTGGTGA 19 2820-88GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 20 TCCAAGGCTCCT 2823-91AGAAAGGCTTGTGATGACACCTGG 21 2823-92 CCAGGTGTCATCACAAGCCTTTCT 22 2823-93AGAAAGGCTGCTTGTGACACCTGG 23 2823-94 CCAGGTGTCACAAGCAGCCTTTCT 24 2823-95TACTGGAAGTGTCTTGGCATCTCC 25 2823-96 GGAGATGCCAAGACACTTCCAGTA 26 2823-97AAGGCACTTTGCATCTCCCCATTC 27 2823-98 GAATGGGGAGATGCAAAGTGCCTT 28 2953-67CTGATGGTCGCAGTTCTAGAT 29 2953-68 ATCTAGAACTGCGACCATCAG 30

Example 2

This example describes the preparation of various TMP-TTR fusions.Several fusion proteins containing TTR and a TMP were prepared. Each ofthe oligonucleotides utilized in this example are listed in Table 2.

A fragment containing the TMP was first amplified from a strainharboring a plasmid encoding a full-length TMP-Fc fusion (see PCTPublication No. 00/24770) using oligonucleotides 2743-96 which encodesthe first 7 codons of the TMP plus a 12 nucleotide 5′ extensionincluding a Nde1 site and 2743-97 which encodes the first 7 codons ofnative TTR and the last 7 codons of the TMP of interest. The resultingPCR fragment was mixed with plasmid derived from strain 5619 and themixture was used as a template for oligonucleotide primers 2743-96 and2693-80 to amplify full-length TMP-TTR. Similar procedures describedabove were used for cloning and expression. The resulting strain,TMP-TTR (strain 5513) had the DNA sequence identified in SEQ ID NO:9.

The TMP was then introduced to the N-terminus of strains 5641, 5642,5643 and 5651, respectively. Plasmid 5513 was digested with Xba1, theresulting Xba1/Xba1 insert containing the TMP and the first 18 codons ofTTR(C10A) was gel purified and ligated with Xba1 restricted, phosphatasetreated and gel purified vector derived from 5641, 5642, 5643 and 5651.DNA sequencing was performed to select the correct orientation for eachfusion. The resulting E. coli strains containing the plasmids aredescribed as follows: TMP-TTR(C10A/A37C)(strain 5704) had the DNAsequence identified in SEQ ID NO:10. TMP-TTR(C10A/D38C)(strain 5705) hadthe DNA sequence identified in SEQ ID NO:11. TMP-TTR(C10A/A81C)(strain5706) had the DNA sequence identified in SEQ ID NO:12.TMP-TTR(C10A/G83C)(strain 5707) had the DNA sequence identified in SEQID NO:13.

TABLE 2 SEQ ID Oligo Sequence Number 2743-96GAGGAATAACATATGATCGAAGGTCCGACTCTGCGT 31 2743-97TTCACCGGTACCAGTTGGACCTGCGCGTGCTGCAAG 32 CCATT

Example 3

This example describes the preparation of PTH (1-34)-TTR(C10A/K15A/G83C)fusion. Each of the oligonucleotides utilized in this example are listedin Table 3.

Two new oligonucleotides, oligonucleotide 2694-01, which encodes thefirst 7 codons of human PTH, and oligonucleotide 2694-03, which encodesthe first 7 codons of TTR and amino acids 28-34 of PTH, were synthesizedto make the fusion. Oligonucleotides 2694-01 and 2694-03 were used in a20-cycle PCR procedure as described above to amplify PTH (1-34) with theTTR linker. The template for this reaction was a strain which harbors aplasmid encoding a PTH1-34-Fc fusion (see PCT Publication No. 01/81415).The resulting PCR mixture was combined with strain 5895 and used as thetemplate to amplify the full length PTH (1-34)-TTR(C10A/K15A/G83C) usingprimers 2694-01 and 2693-80. After sequence confirmation, the resultingexpression strain containing the new plasmid was designatedPTH-TTR(C10A/K15A/G83C)(strain 5920) and had the DNA sequence identifiedin SEQ ID NO:14.

TABLE 3 SEQ ID Oligo Sequence Number 2694-01GAGGAATAACATATGTCTGTTTCTGAAATCCAG 33 2694-03TTCACCGGTACCAGTTGGACCAAAGTTATGAACGTC 34

Example 4

This example describes the preparation of an IL-1ra-TTR(C10A) fusion anda TTR(C10A)-GSGS-IL-1ra fusion. Each of the oligonucleotides utilized inthis example are listed in Table 4.

To prepare the IL-1ra-TTR(C10A) fusion, two oligonucleotides,oligonucleotide 2823-13, which encodes the first 7 codons of the humanprotein IL-1ra, and oligonucleotide 2823-14, which encodes the last 7amino acids of IL-1ra and the first 7 amino acids of TTR, weresynthesized. The plasmid derived from a strain which expresses IL-1ra(see PCT Publication No. 91/08285) was amplified using oligonucleotides2823-13 and 2823-14. The resulting PCR product was mixed with plasmidpurified from strain 5619 and used as a template to amplify full-lengthIL-1-ra-TTR(C10A) using oligonucleotide primers 2823-13 and 2693-80. ThePCR product was cloned, sequenced and expressed as described above. Theresultant strain containing the new plasmid was designatedIL-1ra-TTR(C10A) (strain 5644) and had the DNA sequence identified inSEQ ID NO:15.

To make TTR(C10A)-IL-1ra, the following two oligonucleotides,oligonucleotide 2787-32, which encodes the last 7 amino acids of TTR,the first 7 amino acids of IL-1-ra between which a GSGS linker wasintroduced, and oligonucleotide 2787-33, which encodes the last 7 codonsof IL-1-ra, were synthesized. These two oligonucleotide primers wereused to amplify plasmid 2693, and the resulting PCR product was mixedwith plasmid 5619, and together these were used as a template to amplifyfull-length TTR(C10A)-IL-1ra using primers 2787-33 and 2693-79. The PCRproduct was cloned, sequenced and expressed as described above. Theresultant strain containing the new plasmid was designatedTTR(C10A)-IL-1ra (strain 5645) and had the DNA sequence identified inSEQ ID NO:16.

TABLE 4 SEQ ID Oligo Sequence Number 2823-13GAGGAATAACATATGCGACCGTCCGGACGTAA 35 2823-14TTCTACTTCCAGGAAGACGAAGGTCCAACTGGTACC 36 2787-32GTCGTCACCAATCCCAAGGAAGGTAGTGGTAGCCGA 37 CCGTCCGGCCGTAAGAGC 2787-33CCGCGGATCCTCGAGATTATTCGTCTTCCTGGAAGT 38 AGAA

Example 5

This example describes the preparation of TTR(C10A/G83C)-Bradykinin.Each of the oligonucleotides utilized in this example are listed inTable 5.

Plasmid purified from strain 5651 was used for PCR with oligonucleotideprimer 2693-79 and oligonucleotide primer 2943-47, which is a TTR 3′primer containing a PstI restriction site. This PCR product was gelpurified and restriction digested with NdeI and PstI. The resulting DNAfragment was used in a ligation mixture containing AMG21, digested withNdeI and XhoI, and the annealed oligonucleotide linkers 2943-48, whichencodes the GSGSG linker, and oligonucleotide 2943-49, which encodes theBradykinin antagonist peptide KRPPGFSPL with PstI 5′ and XhoI 3′overlapping ends. GM121 was transformed with this ligation product andDNA was purified from the kanamycin resistant colonies. The DNA sequencewas then confirmed in the resistant colonies. The confirmed strain wasgrown at 30° C. and induced for expression in a 10-liter fermentationdescribed below. The new strain was designated TTR(C10A/G83C)-Bradykinin(strain 5914) and had the DNA sequence identified in SEQ ID NO:17.

TABLE 5 SEQ ID Oligo Sequence Number 2693-79GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 39 2943-47AATATACTGCAGTGGTGGAATAGGAG 40 2943-48GTCGTCACCAATCCCAAGGAAGGATCAGGATCCGGA 41 AAACGTCCGCCGGGTTTCTCCCCGCTGTAATC2943-49 TCGAGATTACAGCGGGGAGAAACCCGGCGGACGTTT 42TCCGGATCCTGATCCTTCCTTGGGATTGGTGACGAC TGCA

Example 6

This example describes the recombinant expression of TTR and the TTRfusion constructs in E. coli. Each of the newly constructed TTR or TTRfusions were first examined for soluble expression at temperaturesranging from 16° C. to 37° C. For this purpose, cultures (25 ml) ofGM221 expressing each of the TTR or TTR fusions were grown in LB mediumsupplemented with 50 μg/ml kanamycin at 37° C. until the optical density(OD) at 600 nm reached 0.5 to 1.0. The cultures were then placed inshakers with temperature settings at 16° C., 20° C., 25° C., 30° C., 34°C. and 37° C., respectively. The induction of gene product expressionfrom the luxPR promoter was achieved following the addition of thesynthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to theculture media to a final concentration of 20 ng/ml. After 6 hours, thebacterial cultures were examined by microscopy for the presence ofinclusion bodies. Often soluble or partial soluble expression could beachieved by growing the cultures at temperatures lower than 30° C. forTTR and its fusions, and this temperature was used for large-scaleexpression. In cases where soluble expression could not be achieved,temperatures at which the level of expression was at the highest wereused for large-scale shakers or fermentors.

Large-scale expression was normally done in 4 liter flasks. Four toeight 4 liter shakers containing 1 liter of LB was inoculated withovernight cultures of TTR or its fusion strains. Expression was doneessentially as described above. Cells were collected by centrifugation.

The fermentation stage, employing aseptic technique, begins with theinoculation from a seed culture of strains produced in a shake flaskcontaining 500 mL of sterilized Luria broth. When this culture obtainedthe appropriate cell density (0.8-2 at 600 nm), the contents were usedto inoculate a 20 liter fermentor containing 10 liter of complex basedgrowth medium. The fermentor is maintained at 30° C. and pH 7 withdissolved oxygen levels kept at 30% saturation. When the cell densityreached an optical density of 10-12 OD units at 600 nm, at which pointthe culture was induced by the addition of N-(3-oxo-hexanoyl) homoserinelactone. At 6 hours post-induction the cells were harvested from thefermentor by centrifugation.

Example 7

This example describes the purification of TTR(C10A/G83C)-Bradykinin.About 193 g of E. coli paste from clone 5914 stored at −80° C. wasdefrosted in 1447 ml of 50 mM tris HCl, 5 mM EDTA, pH 8.0. 50 tablets ofSigma protease inhibitor cocktail 1-873-580 (Saint Louis, Mo.) wasdissolved in the cell suspension and the suspension was passed through amodel 110-Y microfluidizer (Microfluidics, Newton, Mass.) twice at12,000 PSI. The lysate (FIG. 1, Lane 2) was centrifuged at 11,325×g for50 min 4° C. The supernatant was removed as the soluble fraction. Thesoluble fraction was heated in a 65° C. water bath for 30 minutes inpolypropylene bottles, at which time the temperature of the contents was63° C. The soluble fraction was centrifuged at 11,325×g for 50 minutes4° C. The supernatant was removed as Heat Soluble (FIG. 1, Lane 3). Theheat soluble fraction was filtered through a 0.45 μm cellulose acetatefilter with two prefilters and then loaded on to a 240 ml Q-sepharosefast flow (5 cm ID) column (Amersham Pharmacia Biotech, Piscataway,N.J.) at 20 ml/min equilibrated in Q-Buffer A (20 mM tris HCl, 2.5 mMEDTA, pH 8.0) at room temperature (about 23° C.). Column was washed withabout 2300 ml Q-Buffer A at 20 ml/min. Q-column was eluted with a 15column volume linear gradient to 60% Q-Buffer B (20 mM tris HCl, 1 MNaCl, 2.5 mM EDTA, pH 8.0) followed by a 2 column volume step to 100%Q-Buffer B. Fractions containing the TTR fusion as determined bySDS-PAGE were pooled into a single Q-pool (1150 ml) (FIG. 1, Lane 4) and1.77 g of DTT was added. The Q-pool was gently stirred for 30 min atroom temperature (about 23° C.). To the Q-pool, 410 ml of 3.8 M ammoniumsulfate pH 7.0 was slowly added and the pH was lowered from about 7.5 to7.0 by slow addition of 1 M HCl. About one-half of the Q-pool was thenloaded on to an 84 ml phenyl sepharose high performance column (2.6 cmID) (Amersham Pharmacia Biotech) in P-Buffer A (50 mM NaH₂PO₄, 1 Mammonium sulfate, pH 7.0) at 10 ml/min. The column was washed with about170 ml P-Buffer A followed by three step elutions using 50%, 80%, and100% P-Buffer B (50 mM NaH₂PO₄, pH 7.0). The remaining half of theQ-pool was then processed using the same protocol as the first half.Fractions containing the TTR fusion as determined by SDS-PAGE werepooled into a single P-pool (260 ml) (FIG. 1, Lane 5) and the P-pool wasdialyzed against 4 L of HA-Buffer A (10 mM NaH₂PO₄, pH 7.0) for 2 hoursat room temperature (about 23° C.) using 20.4 mm diameter 8 kDa cutoffdialysis tubing (Spectrum Laboratories Inc., Rancho Dominguez, Calif.).The dialysis buffer was changed with a fresh 4 L of HA-Buffer A anddialysis was continued for approximately an additional 15 hours. TheP-pool was removed from dialysis and 600 μl of 1 M DTT was addedfollowed by incubation at room temperature (about 23° C.) for about 1hour. P-pool was loaded on to a 105 ml (2.6 cm) type 1 ceramichydroxyapatite column (Bio-Rad Inc., Hercules, Calif.) at 10 ml/min inHA-Buffer A. Column was washed with approximately 210 ml HA-Buffer A at10 ml/min followed by 4 steps of 12.5%, 25%, 50%, and 100% HA-Buffer B(400 mM NaH₂PO₄, pH 7.0). The flowthrough was pooled as HA-pool (340 ml)(FIG. 1, Lane 6) and 524 mg of DTT was added followed by incubation atroom temperature (about 23° C.) for 1 hour.

About one-half of the HA-pool was loaded on to a 47 ml source 15Q (2.6cm ID) column (Amersham Pharmacia Biotech) at 10 ml/min followed by awash with about 250 ml Q-Buffer A. Column was eluted with a 20 columnvolume linear gradient from 10% to 50% Q-Buffer B followed a step of 2column volumes of 100% Q-Buffer B. The remaining half of the HA-Pool wasthen processed using the same protocol as the first half. Fractionscontaining the TTR fusion as determined by SDS-PAGE were pooled into asingle Q2-pool (260 ml) and concentrated to about 75 ml using a stirredcell with a 10 kDa membrane. Q2-pool (FIG. 1, Lane 7) was then filteredthrough a 0.22 μm cellulose acetate filter and the protein concentrationwas determined to be 16.9 mg/ml using a calculated extinctioncoefficient of 18,450 M⁻¹ cm⁻¹. The pyrogen level was determined to be<1 EU/mg of protein using the Limulus Ameboycyte Lysate assay(Associates of Cape Cod, Falmouth, Mass.). The nucleic acid content wasdetermined to be negligible, since the ratio of the absorbance at 260 nmover 280 nm was determined to be 0.52.

Example 8

This example demonstrates that fusing a peptide to either the C-terminusor N-terminus of TTR(C10A/G83C)does not have a significant impact on itsoligomeric structure. TTR(C10A/G83C), PTH-TTR(C10A/K15A/G83C), andTTR(C10A/G83C)-Bradykinin in 20 mM tris pH 8.0 and about 250 mM NaClwere reduced with 9 mM DTT for about 1 hour at room temperature (about23° C.). About 50 μg of the reduced TTR was injected on to aBiosep-Sec-S 3000 column (7.8 mm ID×300 mm) (Phenomenex, Torrance,Calif.) in SEC-Buffer (50 mM NaH₂PO₄, 500 mM NaCl, pH 6.7) at 1 ml/min.Bio-Rad molecular weight standards (151-1901) were used to calibrate thecolumn and calculate the approximate molecular size of the injectedsamples. As can be seen in FIG. 2, TTR(C10A/G83C) eluted atapproximately 8.8 min corresponding to a molecular size of 49 kDa, whichis comparable to the calculated molecular weight of the tetramer at 55kDa. PTH-TTR(C10A/K15A/G83C) eluted at about 8.6 min corresponding to amolecular size of 67 kDa, which is close to the calculated 71 kDa forthe tetramer. TTR(C10A/G83C)-Bradykinin eluted at about 8.7 mincorresponding to a molecular size of 57 kDa, which is also close to thecalculated 60 kDa for the tetramer.

Example 9

This example demonstrates that fusing a protein containing disulfidebonds to either the C-terminus or N-terminus of TTR(C10A) does not havea significant impact on its oligomeric structure. About 50 μg each ofTTR(C10A), IL-1-ra-TTR(C10A), and TTR(C10A)-IL-1-ra was injected on to aBiosep-Sec-S 3000 column (7.8 mm ID×300 mm) (Phenomenex) in SEC-Bufferat 1 ml/min. Bio-Rad molecular weight standards (151-1901) were used tocalibrate the column and calculate the approximate molecular weight ofthe injected samples. As can be seen in FIG. 3, TTR(C10A) elutes atapproximately 8.8 min, which corresponds to a molecular size of 49 kDawhich is comparable to the calculated molecular weight of the tetramerat 55 kDa. The IL-1-ra-TTR(C10A) fusion eluted at about 7.9 mincorresponding to a molecular size of 188 kDa, which is noticeably largerthan that expected for the tetramer at 124 kDa. Similarly,TTR(C10A)-IL-1-ra eluted at about 7.9 min, again corresponding to amolecular size of 188 kDa compared to the 124 kDa expected for thetetramer. These size discrepancies are likely due to differences in theshape of the molecule, since size exclusion chromatography is shapedependant and the standards are calibrated for globular proteins.

Example 10

This example compares the binding of a TMP sequence fused to thecarboxy-terminus of human immunoglobulin Fc (Fc-TMP) and TMP(m)-TTR tosoluble human myeloproliferative leukemia (MPL) receptor. In addition,this example shows the effect of pegylation of the native TTR cysteineon the binding of the TMP fusion to the MPL receptor. The preparation ofthe pegylated TTR fusions is described in detail in Example 13.

For this example, human MPL receptor was covalently bound to a BIAcoreCM5 chip at R_(L)=1300 R_(U) using the EDC/NHS chemistry as per themanufacturer's instructions (BIAcore, Uppsula, Sweden). All samples werepassed over the chip at 50 μl/min in Dulbecco's PBS (Gibco BRL,Gaithersburg, Md.) with 0.1 mg/ml bovine serum albumin and 0.005% P20(polyoxyethylenesorbitan). The equilibrium endpoint was taken 3 min postinjection. As can be seen in FIG. 4, Fc-TMP shows superior bindingcharacteristics compared to TMP(m)-TTR. Further, this figuredemonstrates that pegylation of the native TTR cysteine (Cys 10)interferes with the binding of TMP to the MPL receptor. The binding ofTMP(m)-TTR-PEG5K showed a significantly repressed binding responsecompared to its non-pegylated counterpart, and TMP(m)-TTR-PEG20K showedan even more severe inhibition. This indicates that the presence of PEGon cysteine 10 likely causes steric interference for binding of thefused TMP to the MPL receptor, and larger PEGs produce moreinterference.

Example 11

This example shows the effect of injecting TMP(m)-TTR into mice on bloodplatelet count. For this example 50 BDF1 mice (Charles RiverLaboratories, Wilmington, Mass.) were split into 5 groups and injected(day 0) subcutaneously with either diluting agent (Dulbecco's PBS with0.1% bovine serum albumin) or diluting agent with 50 μg test protein perkg animal. Each group was divided in half and bled (140 μl) on alternatetime points (day 0, 3, 5, 7, 11, 12, 14, and 17). Mice were anesthetizedwith isoflurane prior to collection.

The collected blood was analyzed for a complete and differential countusing an ADVIA 120 automated blood analyzer with murine software (BayerDiagnostics, New York, N.Y.). As seen in FIG. 5, Fc-TMP showed thegreatest response with platelet count peaking at 4.3×10¹² platelets L⁻¹on day 5, which is over 3.4 times baseline at 1.2×10¹² platelets L⁻¹.TMP(m)-TTR-PEG 5 K was a moderate responder peaking at 2.3×10¹²platelets L⁻¹ which is just under twice the baseline level. Thenon-pegylated form of TMP(m)-TTR shows very little response at 1.5×10¹²platelets L⁻¹ which is only 20% over the baseline level. Thenon-pegylated form of TMP(m)-TTR shows better binding in vitro than itspegylated counterparts (FIG. 4), but it has poor performance in vivocompared to TMP(m)-TTR-PEG 5 K. This indicates that PEG is required toimprove the biological half-life of the TTR construct, and this morethan compensates for the reduced affinity for the receptor.

Example 12

This example demonstrates that mutation of cysteine 10 on TTR to alanineTTR(C10A) does not have a significant impact on its oligomericstructure. About 50 μg each of TTR and TTR(C10A) was injected on to aBiosep-Sec-S 3000 column (7.8 mm ID×300 mm) (Phenomenex) in SEC-Bufferat 1 ml/min. Bio-Rad molecular weight standards (151-1901) were used tocalibrate the column and calculate the approximate molecular size of theinjected samples. As can be seen in FIG. 6, TTR(C10A) elutes atapproximately 8.8 min, which corresponds to a molecular size of 57 kDawhich is similar to the calculated molecular weight of the tetramer at55 kDa. This data combined with the observation that both forms of TTRare resistant to precipitation at 65° C. (data not shown) indicates thatmutation of cysteine 10 to alanine does not have a significant impact onthe structure or stability of TTR.

Example 13

This example demonstrates that mutation of alanine 37 to cysteineTMP-TTR(C10A/A37C), aspartate 38 to cysteine TMP-TTR(C10A/D38C), alanine81 to cysteine TMP-TTR(C10A/A81C), or glycine 83 to cysteineTMP-TTR(C10A/G83C) in a cysteine 10 to alanine background does not havea significant impact on the oligomeric structure of TTR. In addition,this example demonstrates that pegylation of these mutant forms of TTRwith a 5 K or 20 K PEG produces two distinct species of TTR withsignificantly greater molecular size than the unpegylated form. Thepegylation of TTR was carried out by first reducing about 8 ml of theTTR (7.28 mg/ml) with 10 mM DTT for 30 minutes at 30° C. in the presenceof 50 mM tris HCl, pH 8.5. The reduced TTR was then desalted using a 26ml SEPHADEX™ G25 medium column (2.6 cm ID) (Amersham Pharmacia Biotech)at 2.5 ml/min in 20 mM tris HCl, pH 8.5. The concentration was thendetermined by measuring the absorbance of the reduced TTR at 280 nm andusing the calculated extinction coefficient (29,450 M⁻¹ forTMP-TTR(C10A/A37C) (5.14 mg/ml). One-half (4.6 ml) of the reduced samplewas then immediately mixed with 810 μl of 5 mM methoxy-PEG-maleimide 5 K(Shearwater Corporation, Huntsville, Ala.) and the remaining half wasmixed with 1620 μl 2.5 mM methoxy-PEG-maleimide 20 K (ShearwaterCorporation). The reaction was allowed to proceed at 30° C. for 30 minand was quenched by the addition of 46 μl 1 M DTT. Each pegylated samplewas then loaded on to a 5 ml HiTrap Q-sepharose column at 2.5 ml/min andwashed with several column volumes of Q-Buffer A (20 mM tris HCl, pH8.0) at 5 ml/min. The columns were eluted with a linear gradient to 40%Q-Buffer B (20 mM tris HCl, 1 M NaCl, pH 8.0) followed by a 2 columnvolume step to 100% Q-Buffer B. Peak fractions were pooled and theconcentration determined by measuring the absorbance of the pool at 280nm.

About 50 μg of each sample was injected on to a Biosep-Sec-S 3000 column(7.8 mm ID×300 mm) (Phenomenex) in SEC-Buffer at 1 ml/min. Bio-Radmolecular weight standards (151-1901) were used to calibrate the columnand calculate the approximate molecular size of the injected samples. Ascan be seen in FIG. 7, the apparent molecular size of the 4non-pegylated TMP-TTR constructs is between 40 and 45 kDa which isnoticeably lower than the expected 70 kDa tetramer. This retardedelution time is likely due to a slight interaction of the TMP-TTRconstruct with the size exclusion resin, which has been observed withseveral other TMP constructs (data not shown). After conjugation withthe 5 K PEG, the apparent molecular size increases to between 421 and428 kDa (1.53-1.64 minutes more advanced elution than the unpegylatedcounterparts), which is much greater than the expected 90 kDa. Theobservation of an exaggerated molecular weight of pegylated molecules onsize exclusion chromatography is frequently observed phenomenon (datanot shown). The 20 K PEG constructs elute earlier than the largestcalibration standard (670 kDa) showing a 1.28-1.40 minutes more advancedelution than their 5 K pegylated counterparts. This data taken togetherdemonstrates that all 4 engineered mutant forms of TMP-TTR can bepegylated drastically increasing their apparent molecular size.

About 2 μg of the pegylated TMP-TTR constructs were analyzed by SDS-PAGE(FIG. 8). This figure demonstrates by gel shift that most of the TMP-TTRmonomers were modified by only one methoxy-PEG-maleimide, and thereaction was nearly complete leaving very little unmodified monomer.

Example 14

This example demonstrates that Fc-TMP, TMP-TTR(C10A/A37C),TMP-TTR(C10A/D38C), TMP-TTR(C10A/A81C), and TMP-TTR(C10A/G83C) havesimilar affinities for binding human MPL receptor in vitro. For thisexample, Fc-TMP was bound to a BIAcore protein G chip at high density asper the manufacturer's instructions (BIAcore, Uppsula, Sweden). Testproteins were preincubated with 5 nM MPL receptor in Binding Buffer(Dulbecco's PBS (Gibco BRL, Gaithersburg, Md.) with 0.1 mg/ml bovineserum albumin and 0.005% P20 (polyoxyethylenesorbitan) for >2 hours atroom temperature (about 23° C.). For non-pegylated proteins, 0.1 mg/mlheparin was added to prevent non-specific binding. All samples were thenpassed over the chip at 50 μl/min in Binding Buffer. The equilibriumendpoint was taken 3 min post injection. As can be seen in FIG. 9, allTTR constructs showed similar affinity for the MPL receptor withaffinities ranging from 0.881 to 2.333 nm, while the Fc-TMP constructhad affinities ranging from 3.276 to 5.369 nm.

Example 15

This example shows the effect of injecting pegylated TMP-TTR constructsinto mice on blood platelet count. For this example 170 BDF1 mice weresplit into 17 groups and injected (day 0) subcutaneously with 50 μg testprotein per kg animal (TMP fusion construct, Fc-TMP, or a TTR(C10A)control). Each group was divided in half and bled (140 μl) on alternatetime points (day 0, 3, 5, 7, 10, 12, and 14). Mice were anesthetizedwith isoflurane prior to collection.

The collected blood was analyzed for a complete and differential countusing an ADVIA 120 automated blood analyzer with murine software (BayerDiagnostics, New York, N.Y.). As seen in FIG. 10A, Fc-TMP showed thegreatest response with platelet count rising to over 4.2×10¹² plateletsL⁻¹ on day 5 which is 3 times baseline at 1.4×10¹² platelets L⁻¹. All 4of the non-pegylated TMP-TTR constructs preformed better than thecontrol, but not as well as Fc-TMP with platelet counts between 1.8 and2.9×10¹² platelets L⁻¹ on day 5, which is between a 29% and 107%improvement over baseline. As can be seen in FIG. 10B, addition of a 5 KPEG group to the engineered cysteine of all 4 TMP-TTR constructssubstantially improves efficacy with platelet counts between 3.7 and4.4×10¹² platelets L⁻¹ (2.8 to 3.4 times baseline).

Also as can be seen in FIG. 10C, conjugation of a 20 K PEG to TMP-TTRresults in an additional, but less dramatic improvement in efficacy withplatelet counts between 4.2 and 4.6×10¹² platelets L⁻¹ (3.2 to 3.5 timesbaseline). Since all of the TMP fusion constructs had similar bindingaffinities for MPL in vitro, this difference is likely due to the effectof PEG conjugation increasing the effective biological half-life of theconstruct.

Example 16

This example shows the effect of injecting pegylated PTH-TTR constructsinto mice on blood ionized calcium release. For this example 60 male,BDF1, 4 week-old mice were split into 12 groups and injected (day 0)subcutaneously with 8.91 mg test protein per kg animal (PTH fusionconstruct, PTH-Fc, or a TTR(C10A) control). Each group was bled (75 μl)at time points 0, 24, 48, and 72 hours. Mice were anesthetized withisoflurane prior to collection.

The collected blood was analyzed for ionized calcium using aCiba*Corning 634 Ca++/pH analyzer. As seen in FIG. 11, PTH-Fc,PTH-TTR(C10A/K15A/A37C) (PEG 5 K), PTH-TTR(C10A/K15A/A37C) (PEG 20 K),PTH-TTR(C10A/K15A/G83C) (PEG 5 K), and PTH-TTR(C10A/K15A/G83C) (PEG 20K) showed the greatest response with ionized calcium levels risingbetween 2.2 and 2.7 mmol per L at 24 hours post-injection, which is 1.7times baseline at 1.3 mmol per L. At 72 hours post injection, theionized calcium levels of all groups returned to baseline, exceptPTH-TTR(C10A/K15A/A37C) (PEG 5 K), PTH-TTR(C10A/K15A/G83C) (PEG 5 K),and PTH-TTR(C10A/K15A/G83C) (PEG 20 K) treated groups that maintainedelevated ionized calcium levels between 1.8 and 1.9 mmol per L. Thenon-pegylated PTH-TTR constructs were equivalent to or slightly betterthan the TTR(C10A) control at raising serum ionized calcium levels.

Example 17

This example describes the construction of a PTH-TTR(C10A/K15A/A81C)containing plasmid. The Xba1/Xba1 fragment of 5920 was ligated with thepurified vector derived from digesting plasmid 5643 (described inexample 1) with Xba1. The E. coli strain containing the resultingplasmid is described as 5933 PTH-TTRC10A/K15A/A81C.

SEQ ID NO:43: ATGTCTGTTTCTGAAATCCAGCTGATGCATAACCTGGGTAAACATCTGAACTCTATGGAACGTGTTGAATGGCTGCGTAAGAAACTGCAGGACGTTCATAACTTTGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGTGTCTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA

Example 18

This example describes the preparation of a GLP-1-TTR(C10A/G83C) fusionand a GLP-1-TTR(C10A/K15A/G83C) fusion. These constructs were clonedusing plasmid pAMG21, which is described in example 1. Each of theoligonucleotides utilized in this example are listed in Table 6.

The bacterial host GM121 is an E. coli K-12 strain that has beenmodified to contain the lacI^(Q) repressor in the late ebg region (68minutes). The presence of this repressor gene allows the use of thishost with a variety of expression systems, however this repressor isirrelevant to the expression from luxPR. The untransformed host has noantibiotic resistances. Specifically, F'tet/393 was modified by thedelivery of a lacI^(Q) construct into the ebg operon between nucleotideposition 2493 and 2937 as numbered in the Genbank accession numberM64441Gb_Ba with the deletion of the intervening ebg sequence. Theconstruct was delivered to the chromosome using a recombinant phagecalled AGebg-lacI^(Q) #5.

After recombination and resolution only the chromosomal insert describedabove remains in the cell. It was renamed F'tet/GM120. F'tet/GM120 wasthen mutated in the hsdR gene to inactivate it. This was renamedF'tet/GM121. The F'tet episome was cured from the strain, verified astetracyline sensitive and was stored as GM121 (ATCC #202174).

PCR was performed with Roche PCR Core Kit (Cat. No. 1 578 553) in 80 ulreactions containing 2-4 ul mini-prep plasmid DNA template, 1 uM eacholigonucleotide, 0.2 mM each oligonucleotide, 5% DMSO (Sigma), and 2 UTaq DNA polymerase in order to amplify the GLP-1 sequence and a linker.Reaction cycles were 94° C. for 5 min followed by 35 cycles of [94° C.for 20 sec, 45° C. for 30 sec, 72° C. for 1 min]. PCR products werepurified with QIAquick® PCR Purification Kit according to themanufacturer's protocol (QIAGEN). PCR products and vectors were thendigested with NdeI and KpnI (New England Biolabs).

Digested DNA was purified from an agarose gel, then mixed and ligated byT4 DNA ligase (New England Biolabs) for 1.5-2 hours at room temperature.Each ligation mixture was transformed by electroporation into the hoststrain GM121 described above with a Biorad E. coli Pulser at 2.5 KV in acuvette with a gap length of 2 mm. The cells were allowed to recover in2 ml Terrific Broth (TB) for about 3 hours at 37° C. at 250 rpm. 70-100μl of the recovery culture was plated on LB agar containing 40 ug/mlkanamycin. DNA mini-preps were prepared and correct clones were verifiedby nucleotide sequencing.

To prepare the GLP-1-TTR(C10A/G83C) fusion, two oligonucleotides,oligonucleotide 1209-85, which binds the luxR promoter region, and3131-63, which encodes the last 12 amino acids of the fusion linker andthe first 8 amino acids of TTR, were synthesized. A pAMG21 plasmidderived from a strain which expresses a GLP-1 sequence with a N-terminalMet-Lys start followed by a seven Histidine sequence for nickel columnpurification, an Aspartic acid-Glutamic acid-Valine-Aspartic acidsequence for cleavage before the first Histidine of GLP-1 by caspase,the GLP-1 (A2G) sequence, and a 27 amino acid fusion linker wasamplified using oligonucleotides 1209-85 and 3131-63. The PCR productwas cloned and sequenced as described above. The resultant straincontaining the new plasmid was designated GLP-1-TTR(C10A/G83C) (strain6298) and had the DNA sequence identified in SEQ ID NO:47.

To prepare the GLP-1-TTR(C10A/K15A/G83C) fusion, two oligonucleotides,oligonucleotide 3183-83, which contains and NdeI site and encodes thepurification and cleavage sequence described above plus the first sixamino acids of GLP-1 (A2G), and 3183-84, which encodes the last 6 aminoacids of the fusion linker and the first 8 amino acids of TTR, weresynthesized.

A pAMG21 plasmid derived from a strain which expresses a GLP-1 sequencewith a N-terminal Met-Lys start followed by a seven Histidine sequencefor nickel column purification, an Aspartic acid-Glutamicacid-Valine-Aspartic acid sequence for cleavage before the firstHistidine of GLP-1 by caspase, the GLP-1(A2G) sequence, and a 25 aminoacid fusion linker was amplified using oligonucleotides 3183-83 and3183-84. The PCR product was cloned and sequenced as described above.The resultant strain containing the new plasmid was designatedGLP-1-TTR(C10A/K15A/G83C) (strain 6450) and had the DNA sequenceidentified in SEQ ID NO:48.

TABLE 6 SEQ ID Oligo Sequence Number 1209-85 CGTACAGGTTTACGCAAGAAAATGG44 3131-63 GGATTCACCGGTACCAGTTGGACCACCACCACCACC 45ACCACCCGCACTGCCTGAACCAGAGC 3183-83 TGACTAAGCCATATGAAACATCATCACCATCACCAT46 CATGACGAAGTTGATCACGGTGAAGGTACTTTCAC 3183-84GGATTCACCGGTACCAGTTGGACCACCACCACCAC 47 CACCGCTAC SEQ ID NO:48ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTTTCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATTCATCGCTTGGCTGGTTAAAGGTCGTGGTGGTTCTGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGGGTGGTGGTGGTGGTGGTGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA SEQ ID NO:49ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTTTCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATTCATCGCTTGGCTGGTTAAAGGTCGTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGTGGTAGCGGTGGTGGTGGTGGTGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA

Example 19

This example describes the preparation of a GLP-1 (A2G)-K-Fc fusion.This construct was cloned using plasmid pAMG33*, which differs frompAMG21 in that the lux protein and promoters are replaced with lacIbinding sites and an IPTG inducible promoter and the ribosomal bindingsite sequence is shorter (the sequence between the AatII and ClaIrecognition sites is replaced with AATTGTGAGCGGATAACAATTGACAAATGCTAAAATTCTTGATTAATTGTGAGCGGATAACAATTTATCGATTTGATTCTAGAAGGAGGAAT AA)and some of the sequence after the SacII recognition site was deleted(leaving ATAAATAAGTAACGATCCGGTCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCGCAGCAACTTGTCGCGCCAATCGAGCCATGTCGTCGTCAACGACCCCCCATTCAAGAACAGCAAGCAGCATTGAGAACTTTGGAATCCAGTCCCTCTTCCACCTGCTGACCG). Each of the oligonucleotidesutilized in this example are listed in Table 7.

To prepare the GLP-1 (A2G)-Fc fusion, two oligonucleotides,oligonucleotide 2985-92, which contains and NdeI site and encodes thepurification and cleavage sequence described above plus the first eightamino acids of GLP-1 (A2G), and 2687-50, which encodes the amino acids18 through 23 of the Fc, were synthesized. A pAMG33* plasmid derivedfrom a strain which expresses a GLP-1 (A2G) sequence with a N-terminalMet start, a 27 amino acid linker, and an Fc sequence was amplifiedusing oligonucleotides 2985-92 and 2687-50. The PCR product was clonedand sequenced as described above except the enzymes used were NdeI andEcoRI. A colony screening step was included which verified the presenceof insert by PCR with oligonucleotides directed against upstream vectorsequence and the 5 His-Aspartic acid sequence which the insertintroduced. The resultant strain containing the new plasmid wasdesignated GLP-1 (A2G)-K-Fc (strain 5945) and had the DNA sequenceidentified in SEQ ID NO:51.

TABLE 7 SEQ ID Oligo Sequence Number 2985-92AGACCTGTACATATGAAACATCATCACCATCACCAT 50CATGACGAAGTTGATCACGGTGAAGGTACTTTCAC TTCTG 2687-50 GGGGGAAGAGGAAAACTGAC51 SEQ ID NO:52 ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTTTCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATTCATCGCTTGGCTGGTTAAAGGTCGTGGTGGTTCTGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATGGTGGTGGTGGTGGTGACAAAACTCACACATGTCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Example 20

This example describes the cloning of the CH2 domain of animmunoglobulin molecule to the TTR(C10A) to generate TMP-CH2-TTRC10A andTTRC10A-CH2-TMP.

The CH2 domain derived from TMP-Fc was linked to the C-terminal end ofTTR(C10A), i.e., strain 5619, by a two-step PCR procedure. The CH2domain (containing from 5′ to 3′: the last 7 codons of TTR, CH2 and aBamH1-XhoI linker) was first amplified by the following oligos:

2973-77: (SEQ ID NO:53) GTC GTC ACC AAT CCC AAG GAA GGT TCT GGC TCC GGATCA GGG GGA CCG TCA GTT TTC CTC, and 2973-78: (SEQ ID NO:54) CCG CGG ATCCTC GAG ATT AGG ATC CAG AAC CCC CTT TGG CTT TGG AGA TGG T.

This fragment was then fused to 5619 in a subsequent PCR by oligos2973-78 and

2973-79: (SEQ ID NO:55) GAG GAA TAA CAT ATG GGT CCA ACT GGT ACC GGT GAATCC AAG,

followed by Nde1/XhoI digest and cloning into similarly restrictedpAMG21. The resulting plasmid is described as 6017 (TTRC10A-CH2):

SEQ ID NO:56: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGATCCTAA

The Xba1/Xba1 fragment of 6017 was replaced with the correspondingfragment of 5704 as described above to construct TMP-TTRC10A-CH2 (Strain6024):

SEQ ID NO:57: ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGATCCTAA

Construction of TTRC10A-CH2-TMP was done as follows: the TMP fragmentcontaining a 5′ BamHI linker and 3′ XhoI linker was amplified by oligos2694-19 and

2974-70: (SEQ ID NO:58) GAG GAA TAA GGA TCC ATC GAA GGT CCG ACT CTG CG.

The amplified fragment was digested with BamH1 and Xho1 and wassubsequently ligated with similarly restricted 6017. The resulting cloneis described as strain 6104 (TTRC10A-CH2-TMP).

SEQ ID NO:59: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGATCCATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCATAA

Another configuration of this fusion was made as TMP-CH2-TTR2. The CH2domain derived from TMP-Fc was first linked to N-terminus of TTRC10A bya two-step PCR. The CH2 domain (containing from 5′ to 3′: a NdeI-BamHIlinker, CH2 and the first 7 codons of TTR C10A) was first amplified byoligos

2974-65: (SEQ ID NO:60) TTC ACC GGT ACC AGT TGG ACC AGA ACC CCC TTT GGCTTT GGA GAT GGT, and 2974-66: (SEQ ID NO:61) GAG GAA TAA CAT ATG GGA TCCGGT TCT GGG GGA CCG TCA GTT TTC CTC.

This fragment was fused to 5619 in a subsequent PCR by oligos 2974-66and 2693-80 (example 1), followed by restriction with NdeI/XhoI andcloning into similarly restricted pAMG21. The resulting clone isdescribed as 6016 (CH2-TTRC10A):

SEQ ID NO:62: ATGGGATCCGGTTCTGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA

The TMP fragment containing a NdeI linker at 5′ end and a BamHI linkerat 3′ end was amplified by oligos

2974-68: (SEQ ID NO:63) GAG GAA TAA CAT ATG ATC GAA GGT CCG ACT CTG, and2974-69: (SEQ ID NO:64) TAA CAT ATG GGA TCC TGC GCG TGC TGC AAG CCA TTG.

This fragment was then digested with NdeI/BamHI and ligated with thevector which was similarly restricted, gel purified from strain 6016.The resulting clone is described as 6110 (TMP-CH2-TTRC10A):

SEQ ID NO:65: ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCAGGATCCGGTTCTGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA

Example 21

This example describes the construction of TTRC10A/K15A-TMP,TTRC10A/K15A/A81C-TMP and TTRC10A/K15A/G83C-TMP.

TMP was also cloned at the C- termini of TTR and variants thereof. Thefull length TMP containing at its N- terminal end a 5-amino acids linker(gsgsg) plus the last 7 amino acids of wt TTR was amplified by thefollowing set of oligonucleotides in a standard PCR procedure.

2694-18: (SEQ ID NO:66) GTC GTC ACC AAT CCC AAG GAA GGT TCT GGT TCT GGTATC GAA, and 2694-19: (SEQ ID NO:67) CCG CGG ATC CTC GAG ATT ATG CGC GTGCTG CAA GCC ATT G

This PCR fragment was further linked to the 3′ end of wt TTR by a secondPCR utilizing oligos 2694-19 and 2693-79 as described in example 1. Theresulting clone was sequence confirmed and is described as strain 5365(TTR-TMP):

SEQ ID NO:68: ATGGGTCCAACTGGTACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA

The Xba1/Xba1 fragment of 5365 was then replaced by the correspondingXba1/Xba1 fragment of strain 5895 to make strain 5921 (TTRC10A/K15A-TMP)as described above:

SEQ ID NO:69: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA

Plasmid 5921 was subsequently modified by replacing the amino acids atthe following positions: A37, A81 and G83, with the amino acid Cysteineas described in example 1, except that the TTR 3′ oligo utilized withthe mutation oligos (2693-80) in example 1 was replaced with 2694-19,resulting in Strain 5982, containing TTRC10A/K15A/A37C-TMP (SEQ IDNO:70), Strain 5983 containing TTRC10A/K15A/A81C-TMP (SEQ ID NO:71), andStrain 5984 containing TTRC10A/K15A/G83C-TMP (SEQ ID NO:72).

SEQ ID NO:70: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTTGTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA SEQ IDNO:71: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGTGTCTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA SEQ IDNO:72: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAGTGGCTTGCAGC ACGCGCATAA

Example 22

This example describes the construction of TMP-TTRC10A/K15A/A81C andTMP-TTRC10A/K15A/A37C. The Lys at 15th position of TTR was mutagenizedto Ala in strains 5704, 5706 and 5707 by the following methods. Plasmid5513 was digested with Nde1/Kpn1, the insert harboring TMP fragment andthe first 6 amino acids of TTR was purified and ligated with Nde1/Kpn1restricted and gel purified vector derived from strain 5895. Thebacterial strain containing the resulting plasmid is described as 5919(TMP-TTRC10A/K15A/G83C). Plasmid 5919 was then digested with Xba1, theresulting Xba1/Xba1 fragment containing TMP and the first 18 codons ofTTR including the C10A and K15A mutations was gel purified and ligatedwith Xba1 digested, phosphatase treated and gel purified vectors derivedfrom strain 5704 and 5706. The new strains are described as 5918(TMP-TTRC10A/K15A/A81C) and 6023 (TMP-TTRC10A/K15A/A37C).

TMP-TTRC10A/K15A/G83C (SEQ ID NO:73):ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA TMP-TTRC10A/K15A/A81C (SEQID NO:74): ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGTGTCTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA TMP-TTRC10A/K15A/A37C (SEQID NO:75): ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTTGTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA

Example 23

This example describes the expression of GLP-1 fusions proteins in E.coli. 25-100 ml of a saturated overnight culture was used to inoculate50 ml TB with 20 ug/ml kanamycin in a 250 ml baffled flask and incubatedat 37 C, 250 rpm overnight. 10-35 ml of these overnight cultures wereused to inoculate 1 L TB with 20 ug/ml kanamycin in a 2 L baffled flaskand incubated at 37 C, 250 rpm until the optical density at 600 nmreached approximately 0.7. The cultures were then induced to expressrecombinant protein by the addition of: 1 ml of ethanol containing 30ug/ml N-(B-ketocaproyl)-DL-homoserine lactone (Sigma) in the case ofpAMG21, or IPTG to 0.1 mM in the case of pAMG33*. The incubation wascontinued for an additional 2-4 hours and the cells were collected bycentrifugation.

Example 24

This example describes the purification of PTH-TTR(C10A/K15A/A81C).About 197 g of E. coli paste from clone 5933 stored at −80° C. wasdefrosted in 1480 ml of 50 mM tris HCl, 5 mM EDTA, pH 8.0. 60 tablets ofSigma protease inhibitor cocktail 1-873-580 (Saint Louis, Mo.) wasdissolved in the cell suspension and the suspension was passed through amodel 110-Y microfluidizer (Microfluidics, Newton, Mass.) twice at14,000 PSI. The lysate was centrifuged at 11,325×g for 50 min 4° C. Thesupernatant was removed as the soluble fraction. The soluble fractionwas heated in a 65° C. water bath for 30 minutes in polypropylenebottles, at which time the temperature of the contents was 63° C. Thesoluble fraction was centrifuged at 11,325×g for 50 minutes 4° C. Thesupernatant was removed as Heat Soluble. The heat soluble fraction wasfiltered through a 0.45 μm cellulose acetate filter with two prefiltersand then loaded. on to a 240 ml Q-sepharose fast flow (5 cm ID) column(Amersham Pharmacia Biotech, Piscataway, N.J.) at 25 ml/min equilibratedin Q-Buffer A (20 mM tris HCl, 2.5 mM EDTA, pH 8.0) at room temperature(about 23° C.). Column was washed with about 2200 ml Q-Buffer A at 30ml/min. Q-column was eluted with a 15 column volume linear gradient to60% Q-Buffer B (20 mM tris HCl, 1 M NaCl, 2.5 mM EDTA, pH 8.0) followedby a 2 column volume step to 100% Q-Buffer B. Fractions containing theTTR fusion as determined by SDS-PAGE were pooled into a single Q-pool(1300 ml). To the Q-pool, 464 ml of 3.8 M ammonium sulfate pH 7.2 wasslowly added. The solution was centrifuged at 11,325×g for 50 min 4° C.The supernatant was removed as the ammonium sulfate soluble fraction anddiscarded, and the pellet was resuspended in 450 ml 10 mM NaH₂PO₄, pH7.0 by gentle agitation at room temperature for about 30 min. Thesolution was centrifuged at 11,325×g for 50 min 4° C. Supernatant wasremoved as phosphate buffer soluble fraction and filtered through a 0.45μm cellulose acetate filter. Added 240 μl 1 M dithiothreitol to thephosphate buffer soluble fraction and loaded on to a 105 ml (2.6 cm)type 1 ceramic hydroxyapatite column (Bio-Rad Inc., Hercules, Calif.) at10 ml/min in HA-Buffer A. Column was washed with approximately 210 mlHA-Buffer A at 10 ml/min followed by 3 steps of 25%, 50%, and 100%HA-Buffer B (400 mM NaH₂PO₄, pH 7.0). The fractions from the 50% elutionwere pooled as HA-pool (725 ml) and filtered through a 0.22 μm celluloseacetate filter. 1.16 g of dithiothreitol was added to HA-Pool, and thepH was raised to 8.0 using tris base followed by incubation at roomtemperature for about 30 minutes. Diluted HA-pool with 750 ml water andloaded on to a 50 ml source 15Q (2.6 cm ID) column (Amersham PharmaciaBiotech) at 10 ml/min followed by a wash with about 250 ml Q-Buffer A.Column was eluted with a 20 column volume linear gradient from 10% to60% Q-Buffer B followed a step of 2 column volumes of 100% Q-Buffer B.Fractions containing the TTR fusion as determined by SDS-PAGE werepooled into a single Q2-pool (170 ml) and filtered through a 0.22 μmcellulose acetate filter. The protein concentration was determined to be3.7 mg/ml using a calculated extinction coefficient of 23,950 M⁻¹ cm⁻¹.The pyrogen level was determined to be <1 EU/mg of protein using theLimulus Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth,Mass.). The nucleic acid content was determined to be negligible, sincethe ratio of the absorbance at 260 nm over 280 nm was determined to be0.61.

Example 25

This example describes the purification of TMP-TTR(C10A/D38C). About 170g of E. coli paste from clone 5705 stored at −80° C. was defrosted in1275 ml of 50 mM tris HCl, 5 mM EDTA, pH 8.0. 50 tablets of Sigmaprotease inhibitor cocktail 1-873-580 (Saint Louis, Mo.) was dissolvedin the cell suspension and the suspension was passed through a model110-Y microfluidizer (Microfluidics, Newton, Mass.) twice at 14,000 PSI.The lysate was centrifuged at 11,325×g for 30 min 4° C. The supernatantwas removed as the soluble fraction and discarded. The pellets wereresuspended in 1200 ml water using a tissue grinder and 20 more Sigmaprotease inhibitor tablets were added. The suspension was centrifuged at11,325×g for 30 min 4° C. The supernatant was filtered through a WhatmanGF/A filter and 2.1 g of dithiothreitol was added followed by incubationat 7° C. for 30 minutes. The reduced sample was loaded on to a 240 mlQ-sepharose fast flow (5 cm ID) column (Amersham Pharmacia Biotech,Piscataway, N.J.) at 30 ml/min equilibrated in Q-Buffer A (20 mM trisHCl, 0.02% sodium azide, pH 8.0) at 7° C. Column was washed with about1920 ml Q-Buffer A at 30 ml/min. Q-column was eluted with 3 steps of20%, 35%, and 100% Q-Buffer B (20 mM tris HCl, 1 M NaCl, 0.02% sodiumazide, pH 8.0). Added 13 ml 500 mM EDTA pH 8.0 to the flowthrough fromthe Q-column and centrifuged for 30 min at 11,325 g at 4° C. Supernatantwas discarded, and the pellet was resuspended in 700 ml 4 M urea, 20 mMtris HCl, pH 8.0. The urea solublized pellet was then filtered through aWhatman GF/A filter and loaded on to a 240 ml Q-sepharose fast flow (5cm ID) column (Amersham Pharmacia Biotech, Piscataway, N.J.) at 30ml/min equilibrated in Q-Buffer A (20 mM tris HCl, 0.02% sodium azide,pH 8.0) at 7° C. Column was washed with about 1920 ml Q-Buffer A at 30ml/min. Q-column was eluted with 3 steps of 20%, 35%, and 100% Q-BufferB (20 mM tris HCl, 1 M NaCl, 0.02% sodium azide, pH 8.0) at 15 ml/min.Fractions containing the 35% elution peak were pooled, filtered througha 0.22 μm cellulose acetate filter, and 0.5 g of dithiothreitol (10 mMfinal concentration) was added followed by incubation for 30 min at 7°C. The 35% Q-pool was then loaded on to a 45 ml (2.6 cm) type 1 ceramichydroxyapatite column (Bio-Rad Inc., Hercules, Calif.) at 5 ml/min in 20mM tris HCl, 350 mM NaCl, pH 8.0 at 7° C. Column was washed withapproximately 70 ml 20 mM tris HCl, 350 mM NaCl, pH 8.0 at 5 ml/minfollowed by 3 steps of 2.5%, 25%, and 100% HA-Buffer B (400 mM NaH₂PO₄,pH 7.0). The fractions from the 2.5% elution were pooled as HA-pool (80ml) and filtered through a 0.22 μm cellulose acetate filter. The proteinconcentration was determined to be 6.8 mg/ml using a calculatedextinction coefficient of 29,450 M⁻¹ cm⁻¹ . The pyrogen level wasdetermined to be <1 EU/mg of protein using the Limulus Ameboycyte Lysateassay (Associates of Cape Cod, Falmouth, Mass.). The nucleic acidcontent was determined to be negligible, since the ratio of theabsorbance at 260 nm over 280 nm was determined to be 0.54.

Example 26

This example describes the refolding and purification ofTTR(C10A)-CH2-TMP. About 23 g of E. coli paste from clone 6104 stored at−80° C. was defrosted in 200 ml of 50 mM tris HCl, 5 mM EDTA, pH 8.0. 10tablets of Sigma protease inhibitor cocktail 1-873-580 (Saint Louis,Mo.) was dissolved in the cell suspension and the suspension was passedthrough a microfluidizer (Microfluidics, Newton, Mass.) twice at 12,000PSI. The lysate was centrifuged at 15,344×g for 50 min 4° C. Thesupernatant was removed as the soluble fraction and discarded. Thepellet was resuspended in 200 ml 50 mM tris HCl, 5 mM EDTA, pH 8.0 usinga tissue grinder. The suspension was centrifuged at 15,344×g for 50 min4° C. The supernatant was removed as the wash and discarded. The pelletwas resuspended in 50 ml 50 mM tris HCl, 5 mM EDTA, pH 8.0 using atissue grinder. The suspension was centrifuged at 14,000×g for 10 minroom temperature. The supernatant was removed as the wash and discarded.The pellets were dissolved in 50 ml 8 M guanidine HCl, 50 mM tris HCl,pH 8.0 using a sonicator for about 1 min. Dissolved protein was reducedfor 30 min room temperature by adding 500 μl 1 M DTT. Reduced proteinwas centrifuged for 30 min at 20° C. at 27,216 g. Supernatant was thenadded to 4 L 50 mM tris base, 160 mM arginine base, 1 M urea, 1 mMcystamine, 4 mM cysteine, pH 9.5 at 2 ml/min and incubated about 16hours 4° C. Refolded protein was then filtered through a GellmanSUPORCAP® 50 and then concentrated to about 500 ml using a Pall Filtron3 square foot YM10 membrane tangential flow system followed bydiafiltration against 2 L 20 mM tris HCl pH 8.0. Concentrated proteinwas then loaded on to a 45 ml source 15Q (2.6 cm ID) column (AmershamPharmacia Biotech) at 18 ml/min followed by a wash with about 150 mlQ-Buffer A (20 mM tris HCl pH 8.0). Column was eluted with a 20 columnvolume linear gradient from 0% to 60% Q-Buffer B followed a step of 2column volumes of 100% Q-Buffer B. Fractions containing the TTR fusionas determined by SDS-PAGE were pooled into a single Q-pool (29 ml). TheQ-Pool was then concentrated to about 6.3 ml using a MilliporeCENTRIPREP™ 10 and then passed through a Pall ACRODISC® MUSTANG™ Emembrane filter at 1 ml/min. The protein concentration was determined tobe 10.5 mg/ml using a calculated extinction coefficient of 46,410 M⁻¹cm⁻¹ . The pyrogen level was determined to be <1 EU/mg of protein usingthe Limulus Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth,Mass.). The nucleic acid content was determined to be negligible, sincethe ratio of the absorbance at 260 nm over 280 nm was determined to be0.51.

Example 27

This example describes the purification of GLP1-TTR (C10A/K15A/G83C).About 30 g of E. coli paste from clone 6450 stored at −80° C. wasdefrosted in 250 ml of 50 mM NaH₂PO₄, pH 7.0. Cell suspension was passedthrough a microfluidizer (Microfluidics, Newton, Mass.) twice at 12,000PSI. The lysate was centrifuged at 15,344×g for 50 min 4° C. Thesupernatant was discarded as the soluble fraction, and the pellet wasresuspended in 200 ml deoxycholate using a tissue grinder. Thesuspension was centrifuged at 15,344×g for 50 min 4° C. The supernatantwas discarded as the wash, and the pellet was resuspended in 200 mlwater using a tissue grinder. The suspension was centrifuged at 15,344×gfor 50 min 4° C. The supernatant was discarded as the wash, and thepellet was resuspended in 100 ml water using a tissue grinder. Thesuspension was centrifuged at 27,216×g for 30 min room temperature. Thesupernatant was discarded as the wash, and about ⅔ of the pellets weredissolved in 75 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0 byagitation for about 15 min. The suspension was centrifuged at 27,216×gfor 30 min room temperature, and the supernatant was diluted with 18 mlwater. Sample was then loaded on to a 50 ml chelating sepharose fastflow column (Amersham Pharmacia Biotech, Piscataway, N.J.), loaded withNiCl₂, at 5 ml/min. After washing with about 150 ml Ni-Buffer A (6 Mguanidine HCl, 37.5 ml tris HCl, pH 8.0) at 10 ml/min, eluted with twostep of 10% and 100% Ni-Buffer B (6 M guanidine HCl, 37.5 mM tris HCl,400 mM imidazole, pH 8.0). Combined the peak containing the fusionconstruct as Ni-Pool (40 ml) and determined the protein content to be6.4 mg/ml by observing the absorbance at 280 nm in 8 M guanidine HClusing an extinction coefficient of 25,440 M⁻¹. Added 800 μl 500 mM EDTApH 8.0 and removed 80 mg of protein for the PEGylation reaction. Added230 μl 1 M DTT and incubated for 30 min at 30° C. Loaded on to a 130 mlSEPHADEX™ G25 medium column (2.6 cm ID) (Amersham Pharmacia Biotech,Piscataway, N.J.) at 8 ml/min in 20 mM tris HCl, 6 M urea, pH 8.5.Pooled the protein peak as determined by absorbance at 280 nm (22 ml)and determined the concentration to be 3.2 mg/ml by observing theabsorbance at 280 nm in 20 mM tris HCl, 6 M urea, pH 8.5 using anextinction coefficient of 25,440 M−1. Reacted 45% of the bufferexchanged material with 950 μl of 5 mM methoxy-PEG-maleimide 5 K(Shearwater Corporation, Huntsville, Ala.) for 140 min at 30° C. Added100 μl 1 M 2-mercaptoethanol to each reaction to quench. Dialyzedreaction against 1 L 25 mM NaH₂PO₄, 3 M urea, pH 7.25 using a Pierce 10kDa Slidealyzer for 2 hour room temperature. Changed the dialysis bufferfor 25 mM NaH₂PO₄, 10% sucrose, 2 mM EDTA, pH 7.25 and incubated forabout 16 hours room temperature. Added 140 μl 5% CHAPS and 7.28 μl2-mercaptoethanol and 0.475 ml of 3 mg/ml caspase 3 followed by a 2 hourincubation at room temperature. Reaction mixture was loaded on to a 5 mlHiTrap Q-sepharose HP column (Amersham Pharmacia Biotech, Piscataway,N.J.) at 1 ml/min in 20 mM tris HCl pH 8.0 followed by about a 15 mlwash in the same buffer. Column was then developed at 5 ml/min using alinear gradient to 60% 20 mM tris HCl, 1 M NaCl, pH 8.0 followed by astep to 100% of the elution buffer. Fractions containing the TTR fusionas determined by SDS-PAGE were pooled into a single Q-pool (9.5 ml).Concentrated Q-Pool to 3.2 ml using a Millipore CENTRIPREP™ 30 kDa andfiltered through a Pall MUSTANG™ E membrane at about 1 ml/min. DilutedQ-Pool to 6.5 ml with water and added 375 μl acetonitrile. Injected onto a Vydac Protein/Peptide 10×250 mm C₄ column (Vydac, Hisperia, Calif.)in 95% RP-Buffer A (0.1% trifluoroacetic acid) with 5% RP-Buffer B (95%acetonitrile, 0.1% trifluoroacetic acid) at 5 ml/min. Developed columnrunning a linear gradient to 100% RP-Buffer B. Concentrated protein peakto about 3 ml using a Millipore CENTRIPREP™ 30 kDa and diluted to 15 mlusing 20 mM tris HCl pH 8.0. Repeated buffer exchange 3 more times thenpassed though a Pall MUSTANG™ E membrane at about 1 ml/min. The proteinconcentration was determined to be 7.7 mg/ml using a calculatedextinction coefficient of 25,440 M⁻¹ cm⁻¹ . The pyrogen level wasdetermined to be <1 EU/mg of protein using the Limulus Ameboycyte Lysateassay (Associates of Cape Cod, Falmouth, Mass.). The nucleic acidcontent was determined to be negligible, since the ratio of theabsorbance at 260 nm over 280 nm was determined to be 0.54.

Example 28

This example shows the effect of injecting pegylated GLP1-TTR constructsinto mice on blood glucose levels. For this example 40 male, db/db, 9week-old mice were split into 4 groups and injected (hour 0)intraperitoneal with 7.4-16.6 mg test protein per animal (538 pmolmonomers for all groups) (5 K pegylated GLP1-TTR fusion construct 10 mg,20 K pegylated GLP1-TTR fusion construct 10 mg, GLP1-Fc 16.6 mg, and aTTR(C10A) control 7.4 mg). Each group was bled at time points 0(baselinemeasurement), 1, 4, 6, 12, 24, and 48 hours post injection. Food waswithheld from the mice for the first 6 hours of the experiment andreplaced after the bleed at the 6 hour time point.

Each collected drop of blood per time point was analyzed for glucosecontent using a One Touch Profile glucose meter and the results aredepicted in FIG. 12.

Example 29

This example shows the effect of injecting TMP-TTR constructs with fusedantibody CH2 domains into mice on blood platelet count. For this example50 female BDF1 mice were split into 5 groups and injected (day 0)subcutaneously with 50 mg test protein per kg animal (TMP fusionconstruct, Fc-TMP, or a TTR(C10A) control). Each group was divided inhalf and bled (140 ml) on alternate time points (day 0, 3, 5, 7, and10). Mice were anesthetized with isoflurane prior to collection.

The collected blood was analyzed for a complete and differential countusing an ADVIA 120 automated blood analyzer with murine software (BayerDiagnostics, New York, N.Y.). As seen in FIG. 13, Fc-TMP showed thegreatest response with platelet count rising to over 4.2×1012 plateletsL-1 on day 5 which is 3 times baseline at 1.4×1012 platelets L-1. Allthree of the CH2 fused TMP-TTR constructs preformed better than thecontrol, but not as well as Fc-TMP with platelet counts between 2.3×1012and 2.6×1012 platelets L-1 on day 5, which is between a 64% and 86%improvement over baseline.

Example 30

This example shows the effect of injecting pegylated TTR constructs withTMP fused to the carboxy-terminus of pegylated TTR into mice on bloodplatelet count. For this example 80 BDF1 mice were split into 8 groupsand injected (day 0) subcutaneously with 50 mg test protein per kganimal (TMP fusion constructs, Fc-TMP, or a TTR(C10A) control). Eachgroup was divided in half and bled (140 ml) on alternate time points(day 0, 3, 5, 7, 10, and 12). Mice were anesthetized with isofluraneprior to collection.

The collected blood was analyzed for a complete and differential countusing an ADVIA 120 automated blood analyzer with murine software (BayerDiagnostics, New York, N.Y.). As seen in FIG. 14, Fc-TMP and the threeamino terminal (TMP-TTR) fusions showed the greatest response withplatelet count rising between 4.3×10¹² and 4.6×10¹² platelets L-1 on day5 which is over three times baseline at 1.3×10¹² platelets L-1. Allthree of the carboxy terminal (TTR-TMP) constructs performed better thanthe control.

Example 31

This example shows the effect of injecting pegylated TTR-TMP constructscontaining a K15A alteration into mice on blood platelet count. For thisexample 120 BDF1 mice were split into 12 groups and injected (day 0)subcutaneously with 50 mg test protein per kg animal (TMP fusionconstructs, Fc-TMP, or a TTR(C10A) control) (this study was split intotwo batches (PEG 20 K in one and the PEG 5 K and non-pegylated samplesin the other) completed at separate times with repeated controls). Eachgroup was divided in half and bled (140 ml) on alternate time points(day 0, 3, 5, 7, 10, and 12). Mice were anesthetized with isofluraneprior to collection.

The collected blood was analyzed for a complete and differential countusing an ADVIA 120 automated blood analyzer with murine software (BayerDiagnostics, New York, N.Y.). As seen in FIG. 15A, the two non-pegylatedconstructs outperformed the baseline (1.3×10¹² platelets L-1) withplatelet responses at day 5 rising between 1.8×10¹² and 2.0×10¹²platelets L-1. As seen in FIG. 15B, Fc-TMP and the three 5 K pegylatedfusions showed equivalent responses at day 5 with platelet counts risingbetween 3.5×10¹² and 4.4×10¹² platelets L-1 which is at least 2.7 timesbaseline (1.3×10¹² platelets L-1). As seen in FIG. 15C, Fc-TMP and thethree 20 K pegylated fusions showed equivalent responses at day 5 withplatelet count rising between 4.3×10¹² and 4.6×10¹² platelets L-1 whichis over three times baseline at 1.3×10¹² platelets L-1.

In addition, the 20 K pegylated TTR constructs appear to have animproved sustained response with platelets at day 7 ranging from3.7×10¹² to 4.9×10¹² platelets L-1 compared to Fc-TMP at 3.1×10¹²platelets L-1. This sustained response is maintained at day 10 for thethree 20 K pegylated TTR constructs with platelets ranging from 2.3×10¹²to 3.1×10¹² platelets L-1 compared to Fc-TMP at 2.0×10¹² platelets L-1.

1. A transthyretin (TTR) variant comprising the amino acid sequence asset forth in SEQ ID NO:1 having one to three amino acid substitutions,wherein the cysteine at position 10 of SEQ ID NO:1 is substituted foranother amino acid.
 2. The TTR variant of claim 1, wherein the cysteineat position 10 is substituted for alanine.
 3. The TTR variant of claim1, wherein one or more amino acids is substituted to cysteine.
 4. TheTTR variant of claim 3, wherein the amino acid substituted to cysteineis selected from the group consisting of A37, D38, A81, and G83.
 5. TheTTR variant of claim 4, wherein the amino acid substituted to cysteineis A81.
 6. The TTR variant of claim 4, wherein the amino acidsubstituted to cysteine is G83.
 7. The TTR variant of claim 1, whereinone or more lysines are substituted for another amino acid.
 8. The TTRvariant of claim 7, wherein the lysine at position 15 is substituted foranother amino acid.
 9. The TTR variant of claim 8, wherein the lysine atposition 15 is substituted for alanine.
 10. The TTR variant of claim 1,wherein the TTR variant is chemically modified with a chemical selectedfrom the group consisting of dextran, poly(n-vinyl pyurrolidone),polyethylene glycols, propropylene glycol homopolymers, polypropyleneoxide/ethylene oxide co-polymers, polyoxyethylated polyols, andpolyvinyl alcohols.
 11. The TTR variant of claim 10, wherein the TTRvariant is chemically modified with polyethylene glycol (PEG).
 12. TheTTR variant of claim 11, wherein the PEG has a molecular weight ofbetween about 1 kD and about 100 kD.
 13. The TTR variant of claim 12,wherein the PEG has a molecular weight of between about 5 kD and about30 kD.
 14. A fusion protein comprising a biologically active proteinfused to the TTR variant of claim 1, wherein said fusion protein has alonger half-life in serum than the biologically active protein lackingthe TTR variant.
 15. The fusion protein of claim 14, wherein thebiologically active protein is fused to the TTR variant of claim
 10. 16.An isolated nucleic acid encoding the TTR variant of claim
 1. 17. Anexpression vector comprising a nucleic acid sequence encoding the TTRvariant of claim
 1. 18. A host cell comprising the expression vector ofclaim
 16. 19. A method of increasing the half-life in serum of abiologically active protein, said method comprising fusing saidbiologically active protein to a TTR variant of claim
 1. 20. A method ofincreasing the half-life in serum of a biologically active protein, saidmethod comprising fusing said biologically active protein to a TTRvariant of claim 10.